Pharmaceutical Composition

ABSTRACT

The present invention relates to methods and compositions for the therapeutic and diagnostic use in the treatment of diseases and disorders which are caused by or associated with neurofibrillary tangles. In particular, the invention relates to antibodies, which specifically recognize and bind to phosphorylated pathological protein tau-conformers and to methods and compositions involving said antibodies for the therapeutic and diagnostic use in the treatment of tauopathies including Alzheimer&#39;s Disease (AD).

This application is a continuation of U.S. application Ser. No.13/500,608, which has a 371(c) completion date of Apr. 5, 2012, which isthe US national stage of International Application No.PCT/EP2011/067604, filed Oct. 7, 2011, which claims priority to EuropeanApplication No. 11174248.2, filed Jul. 15, 2011, and EuropeanApplication No. 10186810.7, filed Oct. 7, 2010, each of which isincorporated herein by reference in its entirety for any purpose.

REFERENCE TO SEQUENCE LISTING

A sequence listing is submitted concurrently with this application as anASCII formatted text file via EFS-Web, with a file name of“2016-02-17_01147-0001-01US_SeqListing.txt”, a creation date of Feb. 2,2016, and a size of 96,356 bytes. The sequence listing filed via EFS-Webis part of the specification and is hereby incorporated by reference inits entirety.

The present invention is related to methods and compositions for thetherapeutic and diagnostic use in the treatment of diseases anddisorders which are caused by or associated with neurofibrillarytangles. In particular, the invention relates to antibodies, whichspecifically recognize and bind to phosphorylated pathological proteintau-conformers and to methods and compositions involving said antibodiesfor the therapeutic and diagnostic use in the treatment of tauopathiesincluding Alzheimer's Disease (AD).

Neurofibrillary tangles and neuropil threads (NTs) are the majorneuropathological hallmarks of Alzheimer's Disease (AD). They arecomposed of the microtubule-associated protein tau that has undergoneposttranslational modifications; including phosphorylation, deamidationand isomerization on asparaginyl or aspartyl residues. They originate bythe aggregation of hyper-phosphorylated protein tau and its conformers.AD shares this pathology with many neurodegenerative tauopathies, inparticularly with specified types of frontotemporal dementia (FTD).

Protein Tau is a freely soluble, “naturally unfolded” protein that bindsavidly to microtubuli (MT) to promote their assembly and stability. MTsare of major importance for the cytoskeletal integrity of neurons—andthereby for the proper formation and functioning of neuronal circuits,hence for learning and memory. The binding of tau to MT is controlled bydynamic phosphorylation and de-phosphorylation, as demonstrated mainlyin vitro and in non-neuronal cells. Due to the large number of possiblephosphorylation sites (>80), the exact contribution of each and theidentity of the responsible kinases remain largely undefined in vivo.

In AD brain, tau pathology develops later than, and therefore probablyin response to amyloid pathology, which constitutes the essence of theamyloid cascade hypothesis. This is based on and indicated by studies inAD and Down syndrome patients, and is corroborated by studies intransgenic mice with combined amyloid and tau pathology (Lewis et al.,2001; Oddo et al., 2004; Ribe et al., 2005; Muyllaert et al, 2006; 2008;Terwel et al, 2008).

The exact timing of both pathologies in human AD patients as well asmechanisms that link amyloid to tau pathology remain largely unknown,but are proposed to involve activation of neuronal signaling pathwaysthat act on or by GSK3 and cdk5 as the major “tau-kinases” (reviewed byMuyllaert et al, 2006, 2008).

The hypothesis that tauopathy is not an innocent side-effect but a majorpathological executor in AD is based on sound genetic, pathological andexperimental observations that corroborate each other fully:

-   -   in early-onset familial AD cases that are due to mutations in        amyloid protein precursor (APP) or presenilin, the obligate        pathogenic cause is amyloid accumulation, but invariably the        pathology comprises collateral tauopathy, identical to that in        the late-onset sporadic AD cases;    -   severity of cognitive dysfunction and dementia correlates with        tauopathy, not with amyloid pathology, exemplified most recently        by several clinical phase-1&2 studies that include PIB-PET        imaging for amyloid and identify many “false positives”:        cognitively normal individuals with high brain amyloid load;    -   in familial FTD, the tauopathy is provoked by mutant tau and        causes neurodegeneration directly, without amyloid pathology;    -   in experimental mouse models the cognitive defects caused by        amyloid pathology are nearly completely alleviated by the        absence of protein tau (Roberson et al, 2007).

The combined arguments support the hypothesis that protein tau is amajor player in the cognitive demise in AD and related neurodegenerativetauopathies.

A prominent emerging treatment of AD is by passive immunotherapy withspecific mAbs, to clear amyloid peptides and their aggregates that arepresumed to be neuro-toxic or synapto-toxic.

Immunotherapy targeting tau pathology, as proposed here, is anticipatedto counteract the pathological protein tau-conformers that are known orpostulated to cause synaptic dysfunction and neurodegeneration. Amyloidpathology caused and intra-neuronal aggregates of hyper-phosphorylatedprotein tau are proposed to act synergistically in the cognitive anddegenerative cascade of pathological events that lead from mildcognitive impairment (MCI) to the severe dementia of AD. The combinationof tau-directed medication with amyloid-directed (or any other)medication will therefore constitute the preferred and, substantiallymore efficacious treatment of AD, as opposed to current mono-therapy.

Other therapeutic approaches that target protein tau are scarce andcomprise mainly:

-   -   inhibitors of the kinases that are thought to increase the        phosphorylation of tau to pathological levels    -   compounds that block the cytoplasmic aggregation of        hyper-phosphorylated protein tau.

These approaches suffer various draw-backs of specificity and efficacy,a problem they share with attempts to modify the metabolism of APP andamyloid, all emphasizing the importance of a continuous search foradditional treatment options, including immunotherapy against tau.

Practically no efforts have been devoted to define—let alone target—thepathological tau conformers in vivo. In the Aβ42 phase II clinicaltrial, the tangle pathology did not appear to be well considered noranalyzed in much depth (Nicoll et al., 2003; Masliah et al., 2005). Onthe other hand, experimental immunotherapy targeting amyloid in apreclinical mouse model with combined AD-like pathology demonstratedalso an effect on tau pathology although tau aggregates persisted (Oddoet al., 2004).

Some doubts have been cast on the feasibility of approachingintra-cellular protein tau by immunotherapy. These have been counteredby the most recent experimental study in a tauopathy mouse model (Asuniet al., 2007). They showed reduction in tangle pathology and functionalimprovements by vaccination with a protein tau derived phospho-peptide.These data corroborate previous reports of immunotherapy targetingα-synuclein in Parkinson's Disease (PD) and Lewy body disease models(Masliah et al., 2005, 2011) and of superoxide dismutase in anamyotrophic lateral sclerosis (ALS) model (Urushitiani et al., 2007).These diseases are examples wherein intra-cellular proteins lead tosynaptic defects and neurodegeneration by as yet not fully understoodmechanisms. On the other hand, full-length recombinant protein tauproduced in and isolated from bacteria appears not suitable as vaccine,although the adjuvants used, i.e. complete Freunds and pertussis toxin,could have contributed to the negative outcome of that study (Rosenmannet al., 2006).

There is an unmet need for passive and/or active immunotherapies thatwork to counteract the pathological protein conformers that are known—orpresumed—to cause neurodegenerative disorders, such as amyloid pathologyin AD caused, for example, by intra-neuronal aggregates ofhyper-phosphorylated protein tau that are as typical for AD as amyloid.

This unmet need could be met within the scope of the present inventionby providing binding proteins recognizing and binding to majorpathological phospho-epitopes of the tau protein. In particular, thepresent invention provides specific antibodies against linear andconformational, simple and complex phospho-epitopes on protein tau,particularly on aggregated tau protein that are believed to beresponsible for synapto- and neuro-toxicity in tauopathies, includingAD.

Accordingly, the present invention relates in one embodiment to abinding peptide or protein or a functional part thereof, particularly toan antibody, particularly a monoclonal antibody or a functional partthereof, which binding peptide or protein or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to aphospho-epitope on aggregated Tau protein, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes, wherein said binding peptide or antibody has a high bindingaffinity to soluble and insoluble Tau protein, and modulates soluble andinsoluble Tau levels, particularly in the brain, particularly with adissociation constant of at least 10 nM, particularly of at least 8 nM,particularly of at least 5 nM, particularly of at least 2 nM,particularly of at least 1 nM, particularly of at least 500 pM,particularly of at least 400 pM, particularly of at least 300 pM,particularly of at least 200 pM, particularly of at least 100 pM,particularly of at least 50 pM.

In a second embodiment, the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes, wherein said binding peptide or antibody has an associationrate constant of 10⁴ M⁻¹s⁻¹ or greater, particularly of between 3-5×10⁴M⁻¹s⁻¹ or greater, particularly of 10⁵ M⁻¹s⁻¹ or greater; particularlyof 2-9×10⁵ M⁻¹s⁻¹ or greater; particularly of 10⁶ M⁻¹s⁻¹ or greater,particularly of 1-4×10⁶ M⁻¹s⁻¹ or greater, particularly of 10⁷ M⁻¹s⁻¹ orgreater.

In a third embodiment, the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes, wherein said binding peptide or antibody has a high bindingaffinity with a dissociation constant of at least 4 nM and anassociation rate constant of 10⁵ M⁻¹s⁻¹ or greater, particularly adissociation constant of at least 3 nM and an association rate constantof 10⁶ M⁻¹s⁻¹ or greater, particularly a dissociation constant of atleast 2 nM and an association rate constant of 10⁴ M⁻¹s⁻¹ or greater,particularly a dissociation constant of at least 1 nM and an associationrate constant of 10⁵ M⁻¹s⁻¹ or greater, particularly a dissociationconstant of at least 200 pM and an association rate constant of 10⁵M⁻¹s⁻¹ or greater, particularly a dissociation constant of at least 100pM and an association rate constant of 10⁶ M⁻¹s⁻¹ or greater.

One embodiment (4) of the present invention relates to a binding peptideor protein or a functional part thereof, particularly to an antibody,particularly a monoclonal antibody or a functional part thereof,particularly a binding peptide or antibody of any of the precedingembodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody binds to an epitope ona mammalian, particularly on the human Tau protein as shown in SEQ IDNO: 67, selected from the group consisting of Tau aa 15-20 comprising aphosphorylated Tyr at position 18 (Y18), Tau aa 405-412 comprising aphosphorylated Ser at position 409 (pS409), Tau aa 405-411 comprising aphosphorylated Ser at position 409 (pS409); and Tau aa 208-218comprising a phosphorylated Thr at position 212 (pT212) and aphosphorylated Ser at position 214 (pS214).

One embodiment (5) relates to the binding peptide or antibody of any ofthe preceding embodiments, wherein said peptide binds to an epitope on amammalian, particularly on the human Tau protein, but especially thehuman Tau protein as shown in SEQ ID NO: 67, comprising Tau aa 15-20with a phosphorylated Tyr at position 18 (Y18).

One embodiment (6) relates to the binding peptide or antibody of any ofthe preceding embodiments, wherein said peptide binds to an epitope on amammalian, particularly on the human Tau protein, but especially thehuman Tau protein as shown in SEQ ID NO: 67, comprising Tau aa 405-412with a phosphorylated Ser at position 409 (pS409).

One embodiment (7) relates to the binding peptide or antibody of any ofthe preceding embodiments, wherein said peptide binds to an epitope on amammalian, particularly on the human Tau protein, but especially thehuman Tau protein as shown in SEQ ID NO: 67, comprising Tau aa 405-411with a phosphorylated Ser at position 409 (pS409).

One embodiment (8) relates to the binding peptide or antibody of any ofthe preceding embodiments, wherein said peptide binds to an epitope on amammalian, particularly on the human Tau protein, but especially thehuman Tau protein as shown in SEQ ID NO: 67, comprising Tau aa 208-218with a phosphorylated Thr at position 212 (pT212) and a phosphorylatedSer at position 214 (pS214).

In another embodiment (9), the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains, particularly in sequence, a CDR1 with theamino acid sequence shown in SEQ ID NO: 21, 24, 27, 28, 29, 32, 73, 81,93, 101, 106, or an amino acid sequence at least 70%, particularly atleast 80%, particularly at least 85%, particularly at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, particularly atleast 95%, particularly at least 96%, particularly at least 97%,particularly at least 98%, particularly at least 99% or 100% identicalthereto; a CDR2 with the amino acid sequence shown in SEQ ID NO: 22, 25,30, 33, 74, 82, 94, 102, 107, or an amino acid sequence at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, particularly atleast 95%, particularly at least 96%, particularly at least 97%,particularly at least 98%, particularly at least 99% or 100% identicalthereto and a CDR3 with the amino acid sequence shown in SEQ ID NO: 23,26, 31, 34, 75, 83, 95, 103, 108, or an amino acid sequence at least60%, particularly at least 70%, particularly at least 80%, particularlyat least 85%, particularly at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, particularly at least 95%, particularly atleast 96%, particularly at least 97%, particularly at least 98%,particularly at least 99% or 100% identical thereto; and/or a secondbinding domain which contains in sequence a CDR1 with the amino acidsequence shown in SEQ ID NO: 12, 15, 18, 70, 78, 89, 98, or an aminoacid sequence at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, particularly at least 95%, particularly at least 96%,particularly at least 97%, particularly at least 98%, particularly atleast 99% or 100% identical thereto; a CDR2 with the amino acid sequenceshown in SEQ ID NO: 13, 16, 19, 71, 79, 90, 99, 115, or an amino acidsequence at least 80%, particularly at least 85%, particularly at least90%, at least 91%, at least 92%, at least 93%, at least 94%,particularly at least 95%, particularly at least 96%, particularly atleast 97%, particularly at least 98%, particularly at least 99% or 100%identical thereto and a CDR3 with the amino acid sequence shown in SEQID NO: 14, 17, 20, 72, 80, 91, 100, or an amino acid sequence at least60%, at least 70%, at least 80%, particularly at least 85%, particularlyat least 90%, at least 91%, at least 92%, at least 93%, at least 94%,particularly at least 95%, particularly at least 96%, particularly atleast 97%, particularly at least 98%, particularly at least 99% or 100%identical thereto.

One embodiment (10) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains in sequence a CDR1 with the amino acidsequence shown in SEQ ID NO: SEQ ID NO: 21, 24, 27, 28, 29, 32, 73, 81,or an amino acid sequence at least 85% identical thereto; a CDR2 withthe amino acid sequence shown in SEQ ID NO: 22, 25, 30, 33, 74, 82, oran amino acid sequence at least 95% identical thereto and a CDR3 withthe amino acid sequence shown in SEQ ID NO: 23, 26, 31, 34, 75, 83, oran amino acid sequence at least 80% identical thereto; and/or anantibody domain which contains a CDR1 with the amino acid sequence shownin SEQ ID NO: 12, 15, 18, 70, 78, or an amino acid sequence at least 95%identical thereto; a CDR2 with the amino acid sequence shown in SEQ IDNO: 13, 16, 19, 71, 79, or an amino acid sequence at least 85% identicalthereto and a CDR3 with the amino acid sequence shown in SEQ ID NO: 14,17, 20, 72, 80, or an amino acid sequence at least 85% identicalthereto.

One embodiment (11) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains in sequence a CDR1 with the amino acidsequence shown in SEQ ID NO: 21, 24, 27, 28, 29, 32, 73, 81, or an aminoacid sequence at least 90% identical thereto; a CDR2 with the amino acidsequence shown in SEQ ID NO: 22, 25, 30, 33, 74, 82, or an amino acidsequence at least 95% identical thereto and a CDR3 with the amino acidsequence shown in SEQ ID NO: 23, 26, 31, 34, 75, 83, or an amino acidsequence at least 90% identical thereto; and/or a second binding domainwhich contains in sequence a CDR1 with the amino acid sequence shown inSEQ ID NO: 12, 15, 18, 70, 78, or an amino acid sequence at least 95%identical thereto; a CDR2 with the amino acid sequence shown in SEQ IDNO: 13, 16, 19, 71, 79, or an amino acid sequence at least 95% identicalthereto and a CDR3 with the amino acid sequence shown in SEQ ID NO: 14,17, 20, 72, 80, or an amino acid sequence at least 90% identical thereto

One embodiment (12) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain (antibody domain) which contains in sequence a CDR1 withthe amino acid sequence shown in SEQ ID NO: 21, 24, 27, 28, 29, 32, 73,81, or an amino acid sequence at least 90% identical thereto; a CDR2with the amino acid sequence shown in SEQ ID NO: 22, 25, 30, 33, 74, 82,and a CDR3 with the amino acid sequence shown in SEQ ID NO: 23, 26, 31,34, 75, 83, or an amino acid sequence at least 90% identical thereto;and/or a second binding domain which contains in sequence a CDR1 withthe amino acid sequence shown in SEQ ID NO: 12, 15, 18, 70, 78, a CDR2with the amino acid sequence shown in SEQ ID NO: 13, 16, 19, 71, 79, oran amino acid sequence at least 95% identical thereto and a CDR3 withthe amino acid sequence shown in SEQ ID NO: 14, 17, 20, 72, 80, or anamino acid sequence at least 90% identical thereto.

One embodiment (13) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains in sequence a CDR1 with the amino acidsequence shown in SEQ ID NO: 21, 24, 27, 28, 29, 32, or an amino acidsequence at least 98% identical thereto; a CDR2 with the amino acidsequence shown in SEQ ID NO: 22, 25, 30, 33, and a CDR3 with the aminoacid sequence shown in SEQ ID NO: 23, 26, 31, 34, or an amino acidsequence at least 95% identical thereto; and/or a second binding domainwhich contains in sequence a CDR1 with the amino acid sequence shown inSEQ ID NO: 12, 15, 18, a CDR2 with the amino acid sequence shown in SEQID NO: 13, 16, 19, or an amino acid sequence at least 95% identicalthereto and a CDR3 with the amino acid sequence shown in SEQ ID NO: 14,17, 20, or an amino acid sequence at least 90% identical thereto.

One embodiment (14) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains in sequence a CDR1 with the amino acidsequence shown in SEQ ID NO: 21, 24, 27, 28, 29, 32, or an amino acidsequence at least 98% identical thereto; a CDR2 with the amino acidsequence shown in SEQ ID NO: 22, 25, 30, 33, and a CDR3 with the aminoacid sequence shown in SEQ ID NO: 23, 26, 31, 34, or an amino acidsequence at least 98% identical thereto; and/or a second binding domainwhich contains in sequence a CDR1 with the amino acid sequence shown inSEQ ID NO: 12, 15, 18, a CDR2 with the amino acid sequence shown in SEQID NO: 13, 16, 19, or an amino acid sequence at least 98% identicalthereto, and a CDR3 with the amino acid sequence shown in SEQ ID NO: 14,17, 20, or an amino acid sequence at least 90% identical thereto.

One embodiment (15) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains in sequence a CDR1 with the amino acidsequence shown in SEQ ID NO: 21, 24, 27, 28, 29, 32, 73, 81, 93, 101, or106, a CDR2 with the amino acid sequence shown in SEQ ID NO: 22, 25, 30,33, 74, 82, 94, 102, or 107, and a CDR3 with the amino acid sequenceshown in SEQ ID NO: 23, 26, 31, 34, 75, 83, 95, 103, or 108, and/or asecond binding domain which contains in sequence a CDR1 with the aminoacid sequence shown in SEQ ID NO: 12, 15, 18, 70, 78, 89, or 98, a CDR2with the amino acid sequence shown in SEQ ID NO: 13, 16, 19, 71, 79, 90,99, or 115, and a CDR3 with the amino acid sequence shown in SEQ ID NO:14, 17, 20, 72, 80, 91, or 100.

One embodiment (16) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains in sequence a CDR1 with the amino acidsequence shown in SEQ ID NO: 21, or an amino acid sequence at least 76%,particularly at least 80%, particularly at least 85%, particularly atleast 90%, particularly at least 95%, particularly at least 98%,particularly at least 99% identical thereto, a CDR2 with the amino acidsequence shown in SEQ ID NO: 22, or an amino acid sequence at least 95%,particularly 98%, particularly 99% identical thereto, and a CDR3 withthe amino acid sequence shown in SEQ ID NO: 23, or an amino acidsequence at least 66%, particularly at least 70%, particularly at least75%, particularly at least 80%, particularly at least 85%, particularlyat least 90%, particularly at least 95%, particularly at least 98%,particularly at least 99% identical thereto, and/or a second bindingdomain which contains in sequence a CDR1 with the amino acid sequenceshown in SEQ ID NO: 12, or an amino acid sequence at least 95%,particularly 98%, particularly 99% identical thereto, a CDR2 with theamino acid sequence shown in SEQ ID NO: 13, or an amino acid sequence atleast 88%, particularly at least 90%, particularly at least 95%,particularly at least 98%, particularly at least 99% identical thereto,and a CDR3 with the amino acid sequence shown in SEQ ID NO: 14, or anamino acid sequence at least 66%, particularly at least 70%,particularly at least 75%, particularly at least 80%, particularly atleast 85%, particularly at least 90%, particularly at least 95%,particularly at least 98%, particularly at least 99% identical thereto.

One embodiment (17) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains in sequence a CDR1 with the amino acidsequence shown in SEQ ID NO: 24, or SEQ ID NO: 27, or SEQ ID NO: 28, oran amino acid sequence at least 88%, particularly at least 85%,particularly at least 90%, particularly at least 95%, particularly atleast 98%, particularly at least 99% identical thereto, a CDR2 with theamino acid sequence shown in SEQ ID NO: 25, or an amino acid sequence atleast 95%, particularly 98%, particularly 99% and a CDR3 with the aminoacid sequence shown in SEQ ID NO: 26, or an amino acid sequence at least66%, particularly at least 70%, particularly at least 75%, particularlyat least 80%, particularly at least 85%, particularly at least 90%,particularly at least 95%, particularly at least 98%, particularly atleast 99% identical thereto and/or a second binding domain whichcontains in sequence a CDR1 with the amino acid sequence shown in SEQ IDNO: 12, or an amino acid sequence at least 95%, particularly 98%,particularly 99% identical thereto, a CDR2 with the amino acid sequenceshown in SEQ ID NO: 13, or an amino acid sequence at least 88%,particularly at least 90%, particularly at least 95%, particularly atleast 98%, particularly at least 99% identical thereto, and a CDR3 withthe amino acid sequence shown in SEQ ID NO: 14, or an amino acidsequence at least 66%, particularly at least 70%, particularly at least75%, particularly at least 80%, particularly at least 85%, particularlyat least 90%, particularly at least 95%, particularly at least 98%,particularly at least 99% identical thereto.

One embodiment (18) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, according to embodiment (15) comprising a first binding domain,wherein the CDR1 has the amino acid sequence shown in SEQ ID NO: 27, oran amino acid sequence at least 88% identical thereto.

One embodiment (19) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, according to embodiment (15), comprising a first binding domainwherein the CDR1 has the amino acid sequence shown in SEQ ID NO: 28, oran amino acid sequence at least 88% identical thereto.

One embodiment (20) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains in sequence a CDR1 with the amino acidsequence shown in SEQ ID NO: 29, a CDR2 with the amino acid sequenceshown in SEQ ID NO: 30, and a CDR3 with the amino acid sequence shown inSEQ ID NO: 31, or an amino acid sequence at least 95%, particularly 98%,particularly 99% identical thereto and/or a second binding domain whichcontains in sequence a CDR1 with the amino acid sequence shown in SEQ IDNO: 15, or an amino acid sequence at least 95%, particularly 98%,particularly 99% identical thereto, a CDR2 with the amino acid sequenceshown in SEQ ID NO: 16, or an amino acid sequence at least 94%, 95%,96%, 97%, 98%, or 99% identical thereto, and a CDR3 with the amino acidsequence shown in SEQ ID NO: 17, or an amino acid sequence at least 36%,particularly at least 40%, particularly at least 50%, particularly atleast 60%, particularly at least 70%, particularly at least 75%,particularly at least 80%, particularly at least 85%, particularly atleast 90%, particularly at least 95%, particularly at least 98%,particularly at least 99% identical thereto.

One embodiment (21) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes, wherein said binding peptide or antibody comprises a firstbinding domain which contains in sequence a CDR1 with the amino acidsequence shown in SEQ ID NO: 32, a CDR2 with the amino acid sequenceshown in SEQ ID NO: 33, and a CDR3 with the amino acid sequence shown inSEQ ID NO: 34, or an amino acid sequence at least 95%, particularly 98%,particularly 99% identical thereto and/or a second binding domain whichcontains in sequence a CDR1 with the amino acid sequence shown in SEQ IDNO: 18, or an amino acid sequence at least 95%, particularly 98%,particularly 99% identical thereto, a CDR2 with the amino acid sequenceshown in SEQ ID NO: 19, or an amino acid sequence at least 95%, 96%,97%, 98% or 99% identical thereto, and a CDR3 with the amino acidsequence shown in SEQ ID NO: 20, or an amino acid sequence at least 63%,particularly at least 70%, particularly at least 75%, particularly atleast 80%, particularly at least 85%, particularly at least 90%,particularly at least 95%, particularly at least 98%, particularly atleast 99% identical thereto.

One embodiment (22) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains in sequence a CDR1 with the amino acidsequence shown in SEQ ID NO: 73, a CDR2 with the amino acid sequenceshown in SEQ ID NO: 74, and a CDR3 with the amino acid sequence shown inSEQ ID NO: 75, or an amino acid sequence at least at least 60%, at least70%, at least 80%, particularly at least 85%, particularly at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, particularly atleast 95%, particularly at least 96%, particularly at least 97%,particularly at least 98%, particularly at least 99% or 100% identicalthereto and/or a second binding domain which contains in sequence a CDR1with the amino acid sequence shown in SEQ ID NO: 70, or an amino acidsequence at least 95%, particularly 98%, particularly 99% identicalthereto, a CDR2 with the amino acid sequence shown in SEQ ID NO: 71, oran amino acid sequence at least 94%, 95%, 96%, 97%, 98%, or 99%identical thereto, and a CDR3 with the amino acid sequence shown in SEQID NO: 72, or an amino acid sequence at least at least 60%, at least70%, at least 80%, particularly at least 85%, particularly at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, particularly atleast 95%, particularly at least 96%, particularly at least 97%,particularly at least 98%, particularly at least 99% or 100% identicalthereto.

One embodiment (23) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains in sequence a CDR1 with the amino acidsequence shown in SEQ ID NO: 81, a CDR2 with the amino acid sequenceshown in SEQ ID NO: 82, and a CDR3 with the amino acid sequence shown inSEQ ID NO: 83, or an amino acid sequence at least 60%, at least 70%, atleast 80%, particularly at least 85%, particularly at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, particularly atleast 95%, particularly at least 96%, particularly at least 97%,particularly at least 98%, particularly at least 99% or 100% identicalthereto and/or a second binding domain which contains in sequence a CDR1with the amino acid sequence shown in SEQ ID NO: 78, a CDR2 with theamino acid sequence shown in SEQ ID NO: 79, and a CDR3 with the aminoacid sequence shown in SEQ ID NO: 80, or an amino acid sequence at leastat least 60%, at least 70%, at least 80%, particularly at least 85%,particularly at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, particularly at least 95%, particularly at least 96%,particularly at least 97%, particularly at least 98%, particularly atleast 99% or 100% identical thereto.

One embodiment (24) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains in sequence a CDR1 with the amino acidsequence shown in SEQ ID NO: 93, a CDR2 with the amino acid sequenceshown in SEQ ID NO: 94, and a CDR3 with the amino acid sequence shown inSEQ ID NO: 95, or an amino acid sequence at least 60%, at least 70%, atleast 80%, particularly at least 85%, particularly at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, particularly atleast 95%, particularly at least 96%, particularly at least 97%,particularly at least 98%, particularly at least 99% or 100% identicalto any one of the above CDRs and/or a second binding domain whichcontains in sequence a CDR1 with the amino acid sequence shown in SEQ IDNO: 89, a CDR2 with the amino acid sequence shown in SEQ ID NO: 90, anda CDR3 with the amino acid sequence shown in SEQ ID NO: 91, or an aminoacid sequence at least at least 60%, at least 70%, at least 80%,particularly at least 85%, particularly at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, particularly at least 95%,particularly at least 96%, particularly at least 97%, particularly atleast 98%, particularly at least 99% or 100% identical to any one of theabove CDRs.

One embodiment (25) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains in sequence a CDR1 with the amino acidsequence shown in SEQ ID NO: 101, a CDR2 with the amino acid sequenceshown in SEQ ID NO: 102, and a CDR3 with the amino acid sequence shownin SEQ ID NO: 103, or an amino acid sequence at least 60%, at least 70%,at least 80%, particularly at least 85%, particularly at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, particularly atleast 95%, particularly at least 96%, particularly at least 97%,particularly at least 98%, particularly at least 99% or 100% identicalto any one of the above CDRs and/or a second binding domain whichcontains in sequence a CDR1 with the amino acid sequence shown in SEQ IDNO: 98, a CDR2 with the amino acid sequence shown in SEQ ID NO: 99, anda CDR3 with the amino acid sequence shown in SEQ ID NO: 100, or an aminoacid sequence at least at least 60%, at least 70%, at least 80%,particularly at least 85%, particularly at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, particularly at least 95%,particularly at least 96%, particularly at least 97%, particularly atleast 98%, particularly at least 99% or 100% identical to any one of theabove CDRs.

One embodiment (26) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains in sequence a CDR1 with the amino acidsequence shown in SEQ ID NO: 106, a CDR2 with the amino acid sequenceshown in SEQ ID NO: 107, and a CDR3 with the amino acid sequence shownin SEQ ID NO: 108, or an amino acid sequence at least 60%, at least 70%,at least 80%, particularly at least 85%, particularly at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, particularly atleast 95%, particularly at least 96%, particularly at least 97%,particularly at least 98%, particularly at least 99% or 100% identicalto any one of the above CDRs and/or a second binding domain whichcontains in sequence a CDR1 with the amino acid sequence shown in SEQ IDNO: 89, a CDR2 with the amino acid sequence shown in SEQ ID NO: 115, anda CDR3 with the amino acid sequence shown in SEQ ID NO: 91, or an aminoacid sequence at least 60%, at least 70%, at least 80%, particularly atleast 85%, particularly at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, particularly at least 95%, particularly atleast 96%, particularly at least 97%, particularly at least 98%,particularly at least 99% or 100% identical to any one of the aboveCDRs.

In another embodiment (27), the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes, wherein said binding peptide or antibody comprises a firstbinding domain which contains the amino acid sequence shown in SEQ IDNO: 6, 7, 8, 9, 10, 11, or an amino acid sequence at least 90% identicalthereto, and/or a second binding domain which contains the amino acidsequence shown in SEQ ID NO: 1, 2, 3, 4, 5, or an amino acid sequence atleast 85% identical thereto.

In one embodiment (28), the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains the amino acid sequence shown in SEQ IDNO: 6, 7, 8, 9, 10, 11, or an amino acid sequence at least 90% identicalthereto, and/or a second binding domain which contains the amino acidsequence shown in SEQ ID NO: 1, 2, 3, 4, 5, or an amino acid sequence atleast 91% identical thereto.

One embodiment (29) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain (antibody domain) which contains the amino acid sequenceshown in SEQ ID NO: 6, 7, 8, 9, 10, 11, or an amino acid sequence atleast 95% identical thereto; and/or a second binding domain whichcontains the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, 5, oran amino acid sequence at least 91% identical thereto.

In another embodiment (30), the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes, wherein said binding peptide or antibody comprises a firstbinding domain which contains the amino acid sequence shown in SEQ IDNO: 69, 77, 116/92, 97, 105, or an amino acid sequence particularly atleast 85%, particularly at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, particularly at least 95%, particularly atleast 96%, particularly at least 97%, particularly at least 98%,particularly at least 99% or 100% identical thereto, and/or a secondbinding domain which contains the amino acid sequence shown in SEQ IDNO: 68, 76, 88, 96, 104, or an amino acid sequence at least 80%,particularly at least 85%, particularly at least 86%, particularly atleast 87%, particularly at least 88%, particularly at least 89%,particularly at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, particularly at least 95%, particularly at least 96%,particularly at least 97%, particularly at least 98%, particularly atleast 99% or 100% identical thereto.

One embodiment (31) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains the amino acid sequence shown in SEQ IDNO: 6 or SEQ ID NO: 7, or an amino acid sequence at least 90% and 94%,respectively, identical thereto; and/or a second binding domain whichcontains the amino acid sequence shown in SEQ ID NO: 1, or an amino acidsequence at least 91% identical thereto.

One embodiment (32) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains the amino acid sequence shown in SEQ IDNO: 8, or an amino acid sequence at least 95% identical thereto; and/ora second binding domain which contains the amino acid sequence shown inSEQ ID NO: 2, or an amino acid sequence at least 90% identical thereto.

One embodiment (33) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains the amino acid sequence shown in SEQ IDNO: 9, or an amino acid sequence at least 95% identical thereto; and/ora second binding domain which contains the amino acid sequence shown inSEQ ID NO: 3, or an amino acid sequence at least 90% identical thereto.

One embodiment (34) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains the amino acid sequence shown in SEQ IDNO: 10, or an amino acid sequence at least 99% identical thereto; and/ora second binding domain which contains the amino acid sequence shown inSEQ ID NO: 4, or an amino acid sequence at least 89% identical thereto.

One embodiment (35) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains the amino acid sequence shown in SEQ IDNO: 11, or an amino acid sequence at least 98% identical thereto; and/ora second binding domain which contains the amino acid sequence shown inSEQ ID NO: 5, or an amino acid sequence at least 87% identical thereto.

One embodiment (36) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains the amino acid sequence shown in SEQ IDNO: 69, or an amino acid sequence at least 98% or 99% identical thereto;and/or a second binding domain which contains the amino acid sequenceshown in SEQ ID NO: 68, or an amino acid sequence at least 90%, 91%, 92%or 93% identical thereto.

One embodiment (37) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains the amino acid sequence shown in SEQ IDNO: 77, or an amino acid sequence at least 93%, 94% or 95% identicalthereto; and/or a second binding domain which contains the amino acidsequence shown in SEQ ID NO: 76, or an amino acid sequence at least 88%,89%, or 90% identical thereto.

One embodiment (38) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains the amino acid sequence shown in SEQ IDNO: 116, 92, or 118, or an amino acid sequence at least 93%, 94% or 95%identical thereto; and/or a second binding domain which contains theamino acid sequence shown in SEQ ID NO: 88, or an amino acid sequence atleast 90%, 91%, 92% or 93% identical thereto.

One embodiment (39) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains the amino acid sequence shown in SEQ IDNO: 97, or an amino acid sequence at least 99% identical thereto; and/ora second binding domain which contains the amino acid sequence shown inSEQ ID NO: 96, or an amino acid sequence at least 86%, 87%, 88% or 90%identical thereto.

One embodiment (40) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a firstbinding domain which contains the amino acid sequence shown in SEQ IDNO: 105, or an amino acid sequence at least 98%, or 99% identicalthereto; and/or a second binding domain which contains the amino acidsequence shown in SEQ ID NO: 104, or an amino acid sequence at least88%, 89%, or 90% identical thereto.

In another embodiment (41), the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, according to embodiments (22)-(24), wherein said first bindingdomain contains the CDRs as shown in SEQ ID NOs: 21-34 and said secondbinding domain contains the CDRs as shown in SEQ ID NOs: 12-20.

One embodiment (42) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, according to embodiment (31), wherein said first binding domaincontains the CDRs as shown in SEQ ID NOs: 21-23 and SEQ ID NOs: 24-26,respectively, and said second binding domain contains the CDRs as shownin SEQ ID NOs: 12-14.

One embodiment (43) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, according to embodiment (32), wherein said first binding domaincontains the CDRs as shown in SEQ ID NOs: 27, 25, 26 and said secondbinding domain contains the CDRs as shown in SEQ ID NOs: 12-14.

One embodiment (44) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, according to embodiment (33), wherein said first binding domaincontains the CDRs as shown in SEQ ID NOs: 28, 25 and 26, and said secondbinding domain contains the CDRs as shown in SEQ ID NOs: 12-14.

One embodiment (45) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, according to embodiment (34), wherein said first binding domaincontains the CDRs as shown in SEQ ID NOs: 29-31, and said second bindingdomain contains the CDRs as shown in SEQ ID NOs: 15-17.

One embodiment (46) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, according to embodiment (35), wherein said first binding domaincontains the CDRs as shown in SEQ ID NOs: 32-34, and said second bindingdomain contains the CDRs as shown in SEQ ID NOs: 18-20.

One embodiment (47) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, according to embodiment (27), wherein said first binding domaincontains the CDRs as shown in SEQ ID NOs: 73-75, and said second bindingdomain contains the CDRs as shown in SEQ ID NOs: 70-72.

One embodiment (48) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, according to embodiment (27), wherein said first binding domaincontains the CDRs as shown in SEQ ID NOs: 81-83, and said second bindingdomain contains the CDRs as shown in SEQ ID NOs: 78-80.

One embodiment (49) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, according to embodiment (27), wherein said first binding domaincontains the CDRs as shown in SEQ ID NOs: 101-103, and said secondbinding domain contains the CDRs as shown in SEQ ID NOs: 98-100.

One embodiment (50) of the present invention relates to a bindingpeptide or protein or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, according to embodiment (27), wherein said first binding domaincontains the CDRs as shown in SEQ ID NOs: 89, 115, and 91, and saidsecond binding domain contains the CDRs as shown in SEQ ID NOs: 106-108.

In still another embodiment (51), the present invention relates to abinding peptide or protein or a functional part thereof, particularly toan antibody, particularly a monoclonal antibody or a functional partthereof, particularly a binding peptide or antibody of any of thepreceding embodiments, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human Tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody comprises a

-   -   a. first binding domain which contains the amino acid sequence        shown in SEQ ID NO: 6 and/or a second binding domain which        contains the amino acid sequence shown in SEQ ID NO: 1; or a    -   b. first binding domain which contains the amino acid sequence        shown in SEQ ID NO: 7 and/or a second binding domain which        contains the amino acid sequence shown in SEQ ID NO: 1; or a    -   c. first binding domain which contains the amino acid sequence        shown in SEQ ID NO: 8 and/or a second binding domain which        contains the amino acid sequence shown in SEQ ID NO: 2; or a    -   d. first binding domain which contains the amino acid sequence        shown in SEQ ID NO: 9 and/or a second binding domain which        contains the amino acid sequence shown in SEQ ID NO: 3; or a    -   e. first binding domain which contains the amino acid sequence        shown in SEQ ID NO: 10 and/or a second binding domain which        contains the amino acid sequence shown in SEQ ID NO: 4; or a    -   f. first binding domain which contains the amino acid sequence        shown in SEQ ID NO: 11 and/or a second binding domain which        contains the amino acid sequence shown in SEQ ID NO: 5; or a    -   g. first binding domain which contains the amino acid sequence        shown in SEQ ID NO: 69 and/or a second binding domain which        contains the amino acid sequence shown in SEQ ID NO: 68; or a    -   h. first binding domain which contains the amino acid sequence        shown in SEQ ID NO: 77 and/or a second binding domain which        contains the amino acid sequence shown in SEQ ID NO: 76; or a    -   i. first binding domain which contains the amino acid sequence        shown in SEQ ID NO: 116 and/or a second binding domain which        contains the amino acid sequence shown in SEQ ID NO: 88; or a;    -   j. first binding domain which contains the amino acid sequence        shown in SEQ ID NO: 92 and/or a second binding domain which        contains the amino acid sequence shown in SEQ ID NO: 88; or a    -   k. first binding domain which contains the amino acid sequence        shown in SEQ ID NO: 97 and/or a second binding domain which        contains the amino acid sequence shown in SEQ ID NO: 6; or a    -   l. first binding domain which contains the amino acid sequence        shown in SEQ ID NO: 105 and/or a second binding domain which        contains the amino acid sequence shown in SEQ ID NO: 104.

In one embodiment (52) of the invention, the binding peptide of any ofthe preceding embodiments is an antibody, particularly an antibody ofthe IgG2a, IgG2b or the IgG3 isotype, particularly a polyclonalantibody, a monoclonal antibody, a chimeric antibody, a humanizedantibody or a fully human antibody.

One embodiment (48) of the invention relates to a polynucleotideencoding the binding peptide of any one of the preceding embodiments.

In on embodiment (53), said polynucleotide comprises a nucleic acidmolecule selected from the group consisting of

-   -   a. a nucleic acid molecule comprising a nucleotide sequence        encoding a polypeptide comprising the amino acid sequence as        depicted in SEQ ID NOs: 35-45, SEQ ID NOs: 84-87, SEQ ID NO:        109-112 and 117;    -   b. a nucleic acid molecule comprising a nucleotide sequence that        has at least 85% sequence identity to the sequence shown in SEQ        ID NOs: 35-45, SEQ ID NOs: 84-87, SEQ ID NO: 109-112 and 117;    -   c. a nucleic acid molecule comprising a nucleotide sequence that        has at least 90% sequence identity to the sequence shown in SEQ        ID NOs: 35-45, SEQ ID NOs: 84-87, SEQ ID NO: 109-112 and 117;    -   d. a nucleic acid molecule comprising a nucleotide sequence that        has at least 95% sequence identity to the sequence shown in SEQ        ID NOs: 35-45, SEQ ID NOs: 84-87, SEQ ID NO: 109-112 and 117;    -   e. a nucleic acid molecule comprising a nucleotide sequence that        has at least 98% sequence identity to the sequence shown in SEQ        ID NOs: 35-45, SEQ ID NOs: 84-87, SEQ ID NO: 109-112 and 117;    -   f. a nucleic acid molecule comprising a nucleotide sequence that        has at least 99% sequence identity to the sequence shown in SEQ        ID NOs: 35-45, SEQ ID NOs: 84-87, SEQ ID NO: 109-112 and 117;    -   g. a nucleic acid molecule comprising a nucleotide sequence the        complementary strand of which hybridizes to the nucleic acid        molecule of any of a)-f);    -   h. a nucleic acid molecule comprising a nucleotide sequence that        deviates from the nucleotide sequence defined in any of a)-g) by        the degeneracy of the genetic code,

wherein said nucleic acid molecule as defined in any of a)-h) recognizesand specifically binds to a phospho-epitope on a mammalian, particularlyon the human Tau protein or on a fragment thereof, particularly on thehuman Tau protein as shown in SEQ ID NO: 67, selected from the groupconsisting of Tau aa 15-20 comprising a phosphorylated Tyr at position18 (Y18), Tau aa 405-412 comprising a phosphorylated Ser at position 409(pS409), Tau aa 405-411 comprising a phosphorylated Ser at position 409(pS409); and Tau aa 208-218 comprising a phosphorylated Thr at position212 (pT212) and a phosphorylated Ser at position 214 (pS214), Tau aa393-401, comprising a phosphorylated Ser at position 396 (pS396), Tau aa396-401 comprising a phosphorylated Ser at position 396 (pS396), Tau aa394-400 comprising a phosphorylated Ser at position 396 (pS396), Tau aa402-406 comprising a phosphorylated Ser at position 404 (pS404), and Tauaa 393-400 comprising a phosphorylated Ser at position 396 (pS396),wherein, in one embodiment, said binding peptide has a high bindingaffinity with a dissociation constant of at least 10 nM, particularly ofat least 8 nM, particularly of at least 5 nM, particularly of at least 2nM, particularly of at least 1 nM, particularly of at least 500 pM,particularly of at least 400 pM, particularly of at least 300 pM,particularly of at least 200 pM, particularly of at least 100 pM,particularly of at least 50 pM and/or has an association rate constantof 10⁴ M⁻¹s⁻¹ or greater, particularly of between 3-5×10⁴ 5 or greater,particularly of 10⁵ M⁻¹s⁻¹ or greater; particularly of 6-9×10⁵ M⁻¹s⁻¹ orgreater; particularly of 10⁶ M⁻¹s⁻¹ or greater, particularly of 1-4×10⁶M⁻¹s⁻¹ or greater, particularly of 10⁷ M⁻¹s⁻¹ or greater, but, in oneembodiment, does not bind to the corresponding unphosphorylated epitopeand/or to non-related epitopes.

In various embodiments (54) of the invention, a binding peptide isprovided or a functional part thereof, particularly an antibody,particularly a monoclonal antibody or a functional part thereof, or apolynucleotide, according to any one of the preceding embodiments, or acombination thereof, which is capable of specifically recognizing andbinding to a phospho-epitope on a mammalian, particularly on the humanTau protein, particularly a microtubule-associated protein tau,particularly an aggregated microtubule-associated andhyperphosphorylated protein tau such as that present in paired helicalfilaments (PHF), which are the predominant structures in neurofibrillarytangles, neuropil threads and dystrophic neurites, but, in oneembodiment, does not bind to the corresponding unphosphorylated epitopeand/or to non-related epitopes.

In a specific embodiment (55) of the invention, the human tau protein isthe human Tau protein as shown in SEQ ID NO: 67.

The binding peptides and antibodies according to any one of thepreceding embodiments can thus be used (56) for reducing the levels oftotal soluble tau protein, particularly of soluble phosphorylated tauprotein, in the brain, particularly in the brain cortex and/orhippocampus, of a mammal or a human containing increased levels ofsoluble tau protein and/or soluble phosphorylated tau protein.

The binding peptides and antibodies according to any one of thepreceding embodiments can also be used (57) for reducing the levels ofpaired helical filaments containing hyperphosphorylated tau protein(pTau PHF) in the brain, particularly in the brain cortex and/orhippocampus, of a mammal or a human containing increased levels of saidpTau paired helical filaments (pTau PHF).

Reduction of the level of total soluble tau protein and/or solublephosphorylated tau protein and/or pTau paired helical filaments (pTauPHF) in the brain, particularly in the brain cortex and/or hippocampus,of a mammal or a human containing increased levels of said tau proteinvariants, which contribute to tau-protein-associated diseases, disordersor conditions in said mammal or human, may lead to an improvement and/oralleviation of the symptoms associated with such tau-protein-associateddiseases, disorders or conditions (58).

The binding peptides and antibodies according to any one of thepreceding embodiments can therefore be used (59) in therapy,particularly in human therapy, for slowing or halting the progression ofa tau-protein-associated disease, disorder or condition.

The binding peptides and antibodies according to any one of thepreceding embodiments can further be used (60) in therapy, particularlyin human therapy, for improving or alleviating the symptoms associatedwith tau-protein-associated diseases, disorders or conditions such as,for example, impairment or loss of cognitive functions includingreasoning, situational judgement, memory capacity, learning, specialnavigation, etc.

In one embodiment (61), the invention relates to the binding peptidesand antibodies according to any one of the preceding embodiments for usein therapy, particularly for use in the treatment of tauopathies, agroup of tau-protein-associated diseases and disorders, or foralleviating the symptoms associated with tauopathies.

In one embodiment (62), the invention relates to the binding peptidesand antibodies according to any one of the preceding embodiments forretaining or increasing cognitive memory capacity in a mammal sufferingfrom a tauopathy.

In a specific embodiment (63) of the invention, binding peptides andantibodies comprising at least one or all of the light chain CDRs ofantibodies ACI-36-2B6-Ab1, ACI-36-2B6-Ab2, ACI-36-3A8-Ab1, andACI-36-3A8-Ab2 as given in SEQ ID NOs: 25, 26, 27, and SEQ ID NOs: 21,22, 23, respectively, and/or at least one or all of the heavy chain CDRsof antibodies ACI-36-2B6-Ab1, ACI-36-2B6-Ab2, ACI-36-3A8-Ab1, andACI-36-3A8-Ab2 as given in SEQ ID NOs: 12, 13, 14, are used in therapy,particularly in human therapy, for improving or alleviating the symptomsassociated with tau-protein-associated diseases, disorders or conditionssuch as, for example, impairment or loss of cognitive functionsincluding reasoning, situational judgement, memory, learning, specialnavigation, etc.

In another specific embodiment (64) of the invention, the antibodiescomprising the light chain of antibodies ACI-36-2B6-Ab1, ACI-36-2B6-Ab2,ACI-36-3A8-Ab1, and ACI-36-3A8-Ab2 as given in SEQ ID NO: 8 and SEQ IDNOs: 6, 7, respectively, and/or the heavy chain of antibodiesACI-36-2B6-Ab1, ACI-36-2B6-Ab12, ACI-36-3A8-Ab1, and ACI-36-3A8-Ab2 asgiven in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, are used intherapy, particularly in human therapy, for improving or alleviating thesymptoms associated with tau-protein-associated diseases, disorders orconditions such as, for example, impairment or loss of cognitivefunctions including reasoning, situational judgement, memory, learning,special navigation, etc.

In another specific embodiment (65) of the invention, binding peptidesand antibodies comprising at least one or all of the light chain CDRs ofantibodies ACI-33-6C10-Ab1 and ACI-33-6C10-Ab2 as given in SEQ ID NOs:29, 30, 31, and/or at least one or all of the heavy chain CDRs ofantibodies ACI-33-6C10-Ab1 and ACI-33-6C10-Ab2 as given in SEQ ID NOs:15, 16, 17, are used in therapy, particularly in human therapy, forimproving or alleviating the symptoms associated withtau-protein-associated diseases, disorders or conditions such as, forexample, impairment or loss of cognitive functions including reasoning,situational judgement, memory, learning, special navigation, etc.

In another specific embodiment (66) of the invention, binding peptidesand antibodies comprising at least one or all of the light chain CDRs ofantibodies ACI-41-7C2-Ab1 and ACI-41-7C2-Ab2 as given in SEQ ID NOs: 32,33, 34, and/or at least one or all of the heavy chain CDRs of antibodiesACI-41-7C2-Ab1 and ACI-41-7C2-Ab2 as given in SEQ ID NOs: 18, 19, 20,are used in therapy, particularly in human therapy, for improving oralleviating the symptoms associated with tau-protein-associateddiseases, disorders or conditions such as, for example, impairment orloss of cognitive functions including reasoning, situational judgement,memory, learning, special navigation, etc.

In another specific embodiment (67) of the invention, binding peptidesand antibodies comprising at least one or all of the light chain CDRs ofantibodies ACI-35-2A1-Ab1; ACI-35-2A1-Ab2; ACI-35-4A6-Ab1;ACI-35-4A6-Ab2; ACI-35-1D2-Ab1; ACI-35-2G5-Ab1; as given in SEQ ID NOs:73-75, 81-83, 93-95, 101-103, 106-108 and/or at least one or all of theheavy chain CDRs of antibodies ACI-35-2A1-Ab1; ACI-35-2A1-Ab2;ACI-35-4A6-Ab1; ACI-35-4A6-Ab2; ACI-35-1D2-Ab1; ACI-35-2G5-Ab1; as givenin SEQ ID NOs: 70-72, 78-80, 89-91, 98-100, are used in therapy,particularly in human therapy, for improving or alleviating the symptomsassociated with tau-protein-associated diseases, disorders or conditionssuch as, for example, impairment or loss of cognitive functionsincluding reasoning, situational judgement, memory, learning, specialnavigation, etc.

In another specific embodiment (68) of the invention, binding peptidesand antibodies comprising at least one or all of the light chain CDRs ofantibodies ACI-35-2G5-Ab2; ACI-35-2G5-Ab3 as given in SEQ ID NOs:106-108 and/or at least one or all of the heavy chain CDRs of antibodiesACI-35-2G5-Ab2; ACI-35-2G5-Ab3; as given in SEQ ID NOs: 89, 115 and 91,are used in therapy, particularly in human therapy, for improving oralleviating the symptoms associated with tau-protein-associateddiseases, disorders or conditions such as, for example, impairment orloss of cognitive functions including reasoning, situational judgement,memory, learning, special navigation, etc.

Binding of the peptides or antibodies according to the precedingembodiments to tau tangles and pTau on brains may be determined byapplying protein immuno-reactivity testing of selected brain sectionsand by Western blotting of brain homogenates, respectively, as describedin the Examples.

In another embodiment (69), the present invention provides apharmaceutical composition comprising a binding peptide or a functionalpart thereof, particularly an antibody, particularly a monoclonalantibody or a functional part thereof, or a polynucleotide, according toany one of the preceding embodiments, or a combination thereof, in atherapeutically effective amount together with a pharmaceuticallyacceptable carrier.

In one embodiment (70), the binding peptide or a functional partthereof, particularly an antibody, particularly a monoclonal antibody ora functional part thereof, or a polynucleotide, or a pharmaceuticalcomposition, according to any one of the preceding embodiments, or acombination thereof, is used in therapy, particularly in human therapyfor the treatment or alleviation of the symptoms oftau-protein-associated diseases or disorders including neurodegenerativedisorders such as tauopathies.

The binding peptides, antibodies and/or pharmaceutical compositionsaccording to any one of the preceding embodiments may thus be used (71)for slowing or halting the progression of a tau-protein-associateddisease, disorder or condition, upon administration of said bindingpeptides, antibodies and/or pharmaceutical compositions to an animal,particularly a mammal, particularly a human, suffering from such adisease or condition.

The binding peptides, antibodies and/or pharmaceutical compositionsaccording to any one of the preceding embodiments may further be used(72) for improving or alleviating the symptoms associated withtau-protein-associated diseases, disorders or conditions such as, forexample, impairment or loss of cognitive functions including reasoning,situational judgement, memory capacity, learning, special navigation,etc, upon administration of said binding peptides, antibodies and/orpharmaceutical compositions to an animal, particularly a mammal,particularly a human, suffering from such a disease or condition.

In one embodiment (73), the binding peptide or a functional partthereof, particularly an antibody, particularly a monoclonal antibody ora functional part thereof, or a polynucleotide or a pharmaceuticalcomposition, according to any one of the preceding embodiments, or acombination thereof, is used in the treatment of diseases and disorderswhich are caused by or associated with the formation of neurofibrillarylesions, the predominant brain pathology in tauopathy comprising aheterogenous group of neurodegenerative diseases or disorders includingdiseases or disorders which show co-existence of tau and amyloidpathologies including, but not limited to, Alzheimer's Disease,Creutzfeldt-Jacob disease, Dementia pugilistica, Down's Syndrome,Gerstmann-Sträussler-Scheinker disease, inclusion-body myositis, andprion protein cerebral amyloid angiopathy, traumatic brain injury andfurther diseases or disorders which do not show a distinct amyloidpathology including, but not limited to, amyotrophic lateralsclerosis/parkinsonism-dementia complex of Guam, Non-Guamanian motorneuron disease with neurofibrillary tangles, argyrophilic graindementia, corticobasal degeneration, diffuse neurofibrillary tangleswith calcification, frontotemporal dementia with parkinsonism linked tochromosome 17, Hallevorden-Spatz disease, multiple system atrophy,Niemann-Pick disease, type C, Pallido-ponto-nigral degeneration, Pick'sdisease, progressive subcortical gliosis, progressive supranuclearpalsy, Subacute sclerosing panencephalitis, Tangle only dementia,Postencephalitic Parkinsonism, Myotonic dystrophy.

In one embodiment (74), the binding peptide or a functional partthereof, particularly an antibody, particularly a monoclonal antibody ora functional part thereof, or a polynucleotide or a pharmaceuticalcomposition, according to any one of the preceding embodiments, or acombination thereof, is used in the treatment of Alzheimer's Disease.

In one embodiment (75) of the invention, a method is provided formodulating soluble and/or insoluble Tau levels, particularly in thebrain, particularly in the brain cortex and/or hippocampus, of ananimal, particularly a mammal or a human, comprising administering tosaid animal, particularly to said mammal or human, the binding peptideor a functional part thereof, particularly an antibody, particularly amonoclonal antibody or a functional part thereof, or a polynucleotide ora pharmaceutical composition, according to any one of the precedingembodiments, or a combination thereof.

In one aspect, modulation relates to reducing the levels of soluble tauprotein, particularly of soluble phosphorylated tau protein, in thebrain, particularly in the brain cortex and/or hippocampus, of ananimal, particularly a mammal or a human containing increased levels ofsoluble tau protein and/or soluble phosphorylated tau protein.

In one embodiment (76) of the invention, a method is provided forreducing the levels of insoluble tau protein, particularly of pairedhelical filaments containing hyperphosphorylated tau protein (pTau PHF)in the brain, particularly in the brain cortex and/or hippocampus, of ananimal, particularly a mammal or a human, containing increased levels ofinsoluble tau protein, particularly of pTau paired helical filaments(pTau PHF) comprising administering to said animal, particularly to saidmammal or human, the binding peptide or a functional part thereof,particularly an antibody, particularly a monoclonal antibody or afunctional part thereof, or a polynucleotide or a pharmaceuticalcomposition, according to any one of the preceding embodiments, or acombination thereof.

In one embodiment (77), the present invention relates to a method forslowing or halting the progression of a tau-protein-associated disease,disorder or condition in an animal, particularly a mammal or humancomprising administering to said animal, particularly said mammal orhuman, suffering from such a disease or condition, the binding peptideor a functional part thereof, particularly an antibody, particularly amonoclonal antibody or a functional part thereof, or a polynucleotide ora pharmaceutical composition, according to any one of the precedingembodiments, or a combination thereof.

In one embodiment (78), the present invention relates to a method forimproving or alleviating the symptoms associated withtau-protein-associated diseases, disorders or conditions such as, forexample, impairment or loss of cognitive functions including reasoning,situational judgement, memory capacity, learning, special navigation,etc., in an animal, particularly a mammal or a human, comprisingadministering to said animal, particularly to said mammal or human,suffering from such a disease or condition, the binding peptide or afunctional part thereof, particularly an antibody, particularly amonoclonal antibody or a functional part thereof, or a polynucleotide ora pharmaceutical composition, according to any one of the precedingembodiments, or a combination thereof.

In one embodiment (79), the present invention relates to a method forretaining or increasing cognitive memory capacity in a mammal sufferingfrom a tauopathy.

In still another embodiment (80) of the invention, a method is providedfor the treatment of a tau-protein-associated disease or disorderincluding a neurodegenerative disease or disorder such as a tauopathycomprising administering to an animal, particularly to a mammal, butespecially to human, suffering from such a disease or disorder, thebinding peptide or a functional part thereof, particularly an antibody,particularly a monoclonal antibody or a functional part thereof, or apolynucleotide or a pharmaceutical composition, according to any one ofthe preceding embodiments, or a combination thereof.

In one embodiment (81) of the invention, a method is provided for thetreatment of diseases and disorders which are caused by or associatedwith the formation of neurofibrillary lesions, the predominant brainpathology in tauopathy comprising a heterogenous group ofneurodegenerative diseases or disorders including diseases or disorderswhich show co-existence of tau and amyloid pathologies including, butnot limited to, Alzheimer's Disease, Creutzfeldt-Jacob disease, Dementiapugilistica, Down's Syndrome, Gerstmann-Sträussler-Scheinker disease,inclusion-body myositis, and prion protein cerebral amyloid angiopathy,traumatic brain injury and further diseases or disorders which do notshow a distinct amyloid pathology including, but not limited to,amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam,Non-Guamanian motor neuron disease with neurofibrillary tangles,argyrophilic grain dementia, corticobasal degeneration, diffuseneurofibrillary tangles with calcification, frontotemporal dementia withparkinsonism linked to chromosome 17, Hallevorden-Spatz disease,multiple system atrophy, Niemann-Pick disease, type C,Pallido-ponto-nigral degeneration, Pick's disease, progressivesubcortical gliosis, progressive supranuclear palsy, Subacute sclerosingpanencephalitis Tangle only dementia, Postencephalitic Parkinsonism,Myotonic dystrophy, which method comprises administering to an animal,particularly to a mammal, but especially to human, suffering from such adisease or disorder, the binding peptide or a functional part thereof,particularly an antibody, particularly a monoclonal antibody or afunctional part thereof, or a polynucleotide or a pharmaceuticalcomposition according to any one of the preceding embodiments, or acombination thereof.

In another embodiment (82) of the invention, a method is provided forinducing a passive immune response in an animal, particularly a mammalor a human, suffering from a neurodegenerative disorder such astauopathy by administering to said animal or human the binding peptideor a functional part thereof, particularly an antibody, particularly amonoclonal antibody or a functional part thereof, or a polynucleotide,or a pharmaceutical composition, according to any one of the precedingembodiments, or a combination thereof.

In still another embodiment (83) of the invention, a method ofdiagnosing a tau-protein-associated disease, disorder or condition in apatient is provided comprising detecting the immunospecific binding of abinding peptide or an active fragment thereof, particularly an antibody,particularly a monoclonal antibody or a functional part thereof,according to any one of the preceding embodiments, to an epitope of thetau protein in a sample or in situ which includes the steps of

-   -   a. bringing the sample or a specific body part or body area        suspected to contain the tau protein into contact with a binding        peptide or a fragment thereof, particularly an antibody,        particularly a monoclonal antibody or a functional part thereof,        according to any one of the preceding claims, wherein said        binding peptide or antibody or fragment thereof binds an epitope        of the tau protein;    -   b. allowing said binding peptide, particularly said antibody,        particularly said monoclonal antibody or a functional part        thereof, to bind to the tau protein to form an immunological        complex;    -   c. detecting the formation of the immunological complex; and    -   d. correlating the presence or absence of the immunological        complex with the presence or absence of tau protein in the        sample or specific body part or area.

In still another embodiment (84) of the invention, a method fordiagnosing a predisposition to tau-protein-associated disease, disorderor condition in a patient is provided comprising detecting theimmunospecific binding of a binding peptide or an active fragmentthereof, particularly an antibody, particularly a monoclonal antibody ora functional part thereof, according to any one of the precedingembodiments, to an epitope of the tau protein in a sample or in situ,which includes the steps of

-   -   a. bringing the sample or a specific body part or body area        suspected to contain the tau antigen into contact with a binding        peptide or an active fragment thereof, particularly an antibody,        particularly a monoclonal antibody or a functional part thereof,        according to any one of the preceding embodiments, which peptide        or fragment thereof binds an epitope of the tau protein;    -   b. allowing said binding peptide, particularly said antibody,        particularly said monoclonal antibody or a functional part        thereof, to bind to the tau antigen to form an immunological        complex;    -   c. detecting the formation of the immunological complex; and    -   d. correlating the presence or absence of the immunological        complex with the presence or absence of tau antigen in the        sample or specific body part or area;    -   e. comparing the amount of said immunological complex to a        normal control value;

wherein an increase in the amount of said aggregate compared to a normalcontrol value indicates that said patient is suffering from or is atrisk of developing an tau-protein-associated disease or condition.

In one embodiment (85) of the invention, a method is provided formonitoring minimal residual disease in a patient following treatmentwith the binding peptide or a functional part thereof, particularly anantibody, particularly a monoclonal antibody or a functional partthereof, or a polynucleotide, or a pharmaceutical composition, accordingto any one of the preceding embodiments, wherein said method comprises:

-   -   a. bringing the sample or a specific body part or body area        suspected to contain the tau antigen into contact with the        binding peptide or a functional part thereof, particularly an        antibody, particularly a monoclonal antibody or a functional        part thereof, according to any one of the preceding embodiments,        which peptide or fragment thereof binds to an epitope of the tau        protein;    -   b. allowing said binding peptide, particularly said antibody,        particularly said monoclonal antibody or a functional part        thereof, to bind to the tau antigen to form an immunological        complex;    -   c. detecting the formation of the immunological complex; and    -   d. correlating the presence or absence of the immunological        complex with the presence or absence of tau antigen in the        sample or specific body part or area,    -   e. comparing the amount of said immunological complex to a        normal control value,

wherein an increase in the amount of said aggregate compared to a normalcontrol value indicates that said patient still suffers from a minimalresidual disease.

In one embodiment (86), a method is provided for predictingresponsiveness of a patient being treated with the binding peptide or afunctional part thereof, particularly an antibody, particularly amonoclonal antibody or a functional part thereof, or a polynucleotide,or a pharmaceutical composition, according to any one of the precedingembodiments, comprising

-   -   a. bringing the sample or a specific body part or body area        suspected to contain the tau antigen into contact with a binding        peptide or an active fragment thereof, particularly an antibody,        particularly a monoclonal antibody or a functional part thereof        according to any one of the preceding embodiments, which peptide        or fragment thereof binds to an epitope of the tau protein;    -   b. allowing said binding peptide, particularly said antibody,        particularly said monoclonal antibody or a functional part        thereof, to bind to the tau antigen to form an immunological        complex;    -   c. detecting the formation of the immunological complex; and    -   d. correlating the presence or absence of the immunological        complex with the presence or absence of tau antigen in the        sample or specific body part or area,    -   e. comparing the amount of said immunological complex before and        after onset of the treatment,

wherein a decrease in the amount of said aggregate indicates that saidpatient has a high potential of being responsive to the treatment.

In another embodiment (87), the invention relates to a test kit fordetection and diagnosis of tau-protein-associated diseases, disorders orconditions comprising a binding peptide or an active fragment thereof,particularly an antibody, particularly a monoclonal antibody or afunctional part thereof, according to any one of the precedingembodiments.

In one embodiment (88) said test kit comprises a container holding oneor more binding peptides or active fragments thereof, particularly anantibody, particularly a monoclonal antibody or a functional partthereof, according to any one of the preceding embodiments andinstructions for using the binding peptides or antibodies for thepurpose of binding to tau antigen to form an immunological complex anddetecting the formation of the immunological complex such that presenceor absence of the immunological complex correlates with presence orabsence of tau antigen.

In still another embodiment (89), the present invention relates to anepitope selected from the group consisting of Tau aa 15-20 of human tauprotein shown in SEQ ID NO: 67 comprising a phosphorylated Tyr atposition 18 (Y18), Tau aa 405-412 comprising a phosphorylated Ser atposition 409 (pS409), Tau aa 405-411 comprising a phosphorylated Ser atposition 409 (pS409); and Tau aa 208-218 comprising a phosphorylated Thrat position 212 (pT212) and a phosphorylated Ser at position 214(pS214).

In one embodiment (90), said epitope consists of Tau aa 15-20 with aphosphorylated Tyr at position 18 (Y18).

In one embodiment (91), said epitope consists of Tau aa 405-412 with aphosphorylated Ser at position 409 (pS409).

In one embodiment (92), said epitope consists of Tau aa 405-411 with aphosphorylated Ser at position 409 (pS409).

In another embodiment (93), the invention relates to a cell lineproducing a binding peptide or an active fragment thereof, particularlyan antibody, particularly a monoclonal antibody or a functional partthereof according to any one of the preceding embodiments.

In one embodiment (94), the invention relates to a cell line, which ishybridoma cell line 6C10F9C12A11 deposited on Aug. 25, 2010 as DSMACC3079.

In one embodiment (95), the invention relates to a cell line, which ishybridoma cell line 6C10E5E9C12 deposited on Aug. 25, 2010 as DSMACC3081.

In one embodiment (96), the invention relates to a cell line, which ishybridoma cell line 6H1A11C11 deposited on Aug. 25, 2010 as DSM ACC3080.

In one embodiment (97), the invention relates to a cell line, which ishybridoma cell line 6H1G6E6 deposited on Aug. 25, 2010 as DSM ACC3088.

In one embodiment (98), the invention relates to a cell line, which ishybridoma cell line 2B6A10C11 deposited on Aug. 25, 2010 as DSM ACC3084.

In one embodiment (99), the invention relates to a cell line, which ishybridoma cell line 2B6G7A12 deposited on Mar. 10, 2010 as DSM ACC3087.

In one embodiment (100), the invention relates to a cell line, which ishybridoma cell line 3A8A12G7 deposited on Aug. 25, 2010 as DSM ACC3086.

In one embodiment (101), the invention relates to a cell line, which ishybridoma cell line 3A8E12H8 deposited on Aug. 25, 2010 as DSM ACC3085.

In one embodiment (102), the invention relates to a cell line, which ishybridoma cell line 7C2(1)F10C10D3 deposited on Aug. 25, 2010 as DSMACC3082.

In one embodiment (103), the invention relates to a cell line, which ishybridoma cell line 7C2(2)B9F11D5 deposited on Aug. 25, 2010 as DSMACC3083.

In one embodiment (103a), the invention relates to a cell line, which ishybridoma cell line A4-4A6-48 deposited on Aug. 30, 2011 as DSM ACC3136.

In one embodiment (103b), the invention relates to a cell line, which ishybridoma cell line A6-2G5-30 deposited on Aug. 30, 2011 as DSM ACC3137.

In one embodiment (103c), the invention relates to a cell line, which ishybridoma cell line A6-2G5-41 deposited on Aug. 30, 2011 as DSM ACC3138.

In one embodiment (103d), the invention relates to a cell line, which ishybridoma cell line A4-2A1-18 deposited on Aug. 30, 2011 as DSM ACC3139.

In one embodiment (103e), the invention relates to a cell line, which ishybridoma cell line A4-2A1-40 deposited on Aug. 30, 2011 as DSM ACC3140.

In one embodiment (103e), the invention relates to a cell line, which ishybridoma cell line A6-1D2-12 deposited on Sep. 6, 2011 as DSM ACC3141.

In one embodiment (104), the invention relates to a monoclonal antibodyor a functional part thereof comprising a light chain (VL) and/or aheavy chain (VH) domain, which is encoded by a polynucleotide located ona nucleotide fragment that can be obtained by PCR amplification of DNAof hybridoma cell line 6C10F9C12A11 deposited on Aug. 25, 2010 as DSMACC3079 using

-   -   a. a primer pair comprising a 5′-primer of SEQ ID NO: 54 and a        3′-primer of SEQ ID NO: 51 for amplification of a first binding        domain; and/or    -   b. a mix of primers comprising a 5′-primer of SEQ ID NO: 53 and        SEQ ID NO: 54 and a 3′-primer of SEQ ID NO: 47 for amplification        of a second binding domain.

In one embodiment (105), the invention relates to a monoclonal antibodyor a functional part thereof comprising a light chain (VL) and/or aheavy chain (VH) domain, which is encoded by a polynucleotide located ona nucleotide fragment that can be obtained by PCR amplification of DNAof hybridoma cell line 6C10E5E9C12 deposited on Aug. 25, 2010 as DSMACC3081 using

-   -   a. a mix of primers comprising a 5′-primer of SEQ ID NO: 48 and        SEQ ID NO: 49 and a 3′-primer of SEQ ID NO: 51 for amplification        of a first binding domain; and/or    -   b. a mix of primers comprising a 5′-primer of SEQ ID NO: 53 and        SEQ ID NO: 54 and a 3′-primer of SEQ ID NO: 47 for amplification        of a second binding domain.

In one embodiment (106), the invention relates to a monoclonal antibodyor a functional part thereof comprising a light chain (VL) and/or aheavy chain (VH) domain, which is encoded by a polynucleotide located ona nucleotide fragment that can be obtained by PCR amplification of DNAof hybridoma cell line 6H1A11C11 deposited on Aug. 25, 2010 as DSMACC3080 using

-   -   a. a primer pair comprising a 5′-primer of SEQ ID NO: 50 and a        3′-primer of SEQ ID NO: 51 for amplification of a first binding        domain; and/or    -   b. a primer pair comprising a 5′-primer of SEQ ID NO: 46 and a        3′-primer of SEQ ID NO: 47 for amplification of a second binding        domain.

In one embodiment (107), the invention relates to a monoclonal antibodyor a functional part thereof comprising a light chain (VL) and/or aheavy chain (VH) domain, which is encoded by a polynucleotide located ona nucleotide fragment that can be obtained by PCR amplification of DNAof hybridoma cell line 6H1G6E6 deposited on Aug. 25, 2010 as DSM ACC3088using

-   -   a. a primer pair comprising a 5′-primer of SEQ ID NO: 50 and a        3′-primer of SEQ ID NO: 51 for amplification of a first binding        domain; and/or    -   b. a primer pair comprising a 5′-primer of SEQ ID NO: 46 and a        3′-primer of SEQ ID NO: 47 for amplification of a second binding        domain.

In one embodiment (108), the invention relates to a monoclonal antibodyor a functional part thereof comprising a light chain (VL) and/or aheavy chain (VH) domain, which is encoded by a polynucleotide located ona nucleotide fragment that can be obtained by PCR amplification of DNAof hybridoma cell line 2B6A10C11 deposited on Aug. 25, 2010 as DSMACC3084 using

-   -   a. a primer pair comprising a 5′-primer of SEQ ID NO: 50 and a        3′-primer of SEQ ID NO: 51 for amplification of a first binding        domain; and/or    -   b. a mix of primers comprising a 5′-primer of SEQ ID NO: 46 and        SEQ ID NO: 52 and a 3′-primer of SEQ ID NO: 47 for amplification        of a second binding domain.

In one embodiment (109), the invention relates to a monoclonal antibodyor a functional part thereof comprising a light chain (VL) and/or aheavy chain (VH) domain, which is encoded by a polynucleotide located ona nucleotide fragment that can be obtained by PCR amplification of DNAof hybridoma cell line 2B6G7A12 deposited on Aug. 25, 2010 as DSMACC3087 using

-   -   a. a primer pair comprising a 5′-primer of SEQ ID NO: 50 and a        3′-primer of SEQ ID NO: 51 for amplification of a first binding        domain; and/or    -   b. a mix of primers comprising a 5′-primer of SEQ ID NO: 46 and        SEQ ID NO: 52 and a 3′-primer of SEQ ID NO: 47 for amplification        of a second binding domain.

In one embodiment (110), the invention relates to a monoclonal antibodyor a functional part thereof comprising a light chain (VL) and/or aheavy chain (VH) domain, which is encoded by a polynucleotide located ona nucleotide fragment that can be obtained by PCR amplification of DNAof hybridoma cell line 3A8A12G7 deposited on Aug. 25, 2010 as DSMACC3086 using

-   -   a₁. a mix of primers comprising a 5′-primer of SEQ ID NO: 48 and        SEQ ID NO: 49 and a 3′-primer of SEQ ID NO: 51 for amplification        of a first binding domain; or    -   a₂. a primer pair comprising a 5′-primer of SEQ ID NO: 50 and a        3′-primer of SEQ ID NO: 51 for amplification of a first binding        domain; and/or    -   b. a primer pair comprising a 5′-primer of SEQ ID NO: 46 and a        3′-primer of SEQ ID NO: 47 for amplification of a second binding        domain.

In one embodiment (111), the invention relates to a monoclonal antibodyor a functional part thereof comprising a light chain (VL) and/or aheavy chain (VH) domain, which is encoded by a polynucleotide located ona nucleotide fragment that can be obtained by PCR amplification of DNAof hybridoma cell line 3A8E12H8 deposited on Aug. 25, 2010 as DSMACC3085 using

-   -   a₁. a mix of primers comprising a 5′-primer of SEQ ID NO: 48 and        SEQ ID NO: 49 and a 3′-primer of SEQ ID NO: 51 for amplification        of a first binding domain; or    -   a₂. a primer pair comprising a 5′-primer of SEQ ID NO: 50 and a        3′-primer of SEQ ID NO: 51 for amplification of a first binding        domain; and/or    -   b. a primer pair comprising a 5′-primer of SEQ ID NO: 46 and a        3′-primer of SEQ ID NO: 47 for amplification of a second binding        domain.

In one embodiment (112), the invention relates to a monoclonal antibodyor a functional part thereof comprising a light chain (VL) and/or aheavy chain (VH) domain, which is encoded by a polynucleotide located ona nucleotide fragment that can be obtained by PCR amplification of DNAof hybridoma cell line 7C2(1)F10C10D3 deposited on Aug. 25, 2010 as DSMACC3082 using

-   -   a. a mix of primers comprising a 5′-primer of SEQ ID NO: 49; SEQ        ID NO: 56 and SEQ ID NO: 57 and a 3′-primer of SEQ ID NO: 51 for        amplification of a first binding domain;    -   b. a mix of primers comprising a 5′-primer of SEQ ID NO: 53 and        SEQ ID NO: 55 and a 3′-primer of SEQ ID NO: 47 for amplification        of a second binding domain.

In one embodiment (113), the invention relates to a monoclonal antibodyor a functional part thereof comprising a light chain (VL) and/or aheavy chain (VH) domain, which is encoded by a polynucleotide located ona nucleotide fragment that can be obtained by PCR amplification of DNAof hybridoma cell line 7C2(2)B9F11D5 deposited on Aug. 25, 2010 as DSMACC3083 using

-   -   a. a pair of primers comprising a 5′-primer of SEQ ID NO: 57 and        a 3′-primer of SEQ ID NO: 51 for amplification of a first        binding domain;    -   b. a mix of primers comprising a 5′-primer of SEQ ID NO: 53 and        SEQ ID NO: 55 and a 3′-primer of SEQ ID NO: 47 for amplification        of a second binding domain.

In one embodiment (114), the invention relates to a monoclonal antibodyor a functional part thereof comprising a light chain (VL) and/or aheavy chain (VH) domain, which is encoded by a polynucleotide located ona nucleotide fragment that can be obtained by PCR amplification of DNAof hybridoma cell line A4-2A1-18 deposited on Aug. 30, 2011 as DSMACC3139 using

-   -   a. a primer pair comprising a 5′-primer of SEQ ID NO: 149 and a        3′-primer of SEQ ID NO: 51 for amplification of a first binding        domain; and/or    -   b. a mix of primers comprising a 5′-primer selected from the        group consisting of SEQ ID NOs: 120, 123, 124, 136, 137, 138,        139, and 140 and a 3′-primer selected from the group consisting        of SEQ ID NOs: 131, 134, and 141-148, for amplification of a        second binding domain.

In one embodiment (115), the invention relates to a monoclonal antibodyor a functional part thereof comprising a light chain (VL) and/or aheavy chain (VH) domain, which is encoded by a polynucleotide located ona nucleotide fragment that can be obtained by PCR amplification of DNAof hybridoma cell line A6-2G5-30 deposited on Aug. 30, 2011 as DSMACC3137 using

-   -   a. a mix of primers comprising a 5′-primer selected from the        group consisting of SEQ ID NOs: 51 and 169-174 and a 3′-primer        of SEQ ID NO: 51, for amplification of a first binding domain;        and/or    -   b. a mix of primers comprising a 5′-primer selected from the        group consisting of SEQ ID NOs: 124, 127, and 150-158 and a        3′-primer selected from the group consisting of SEQ ID NOs: 130,        and 159-168, for amplification of a second binding domain.

In one embodiment (116), the invention relates to a monoclonal antibodyor a functional part thereof comprising a light chain (VL) and/or aheavy chain (VH) domain, which is encoded by a polynucleotide located ona nucleotide fragment that can be obtained by PCR amplification of DNAof hybridoma cell line A4-2A1-40 deposited on Aug. 30, 2011 as DSMACC3140 using

-   -   a. a mix of primers comprising a 5′-primer selected from the        group consisting of SEQ ID NOs: 178, 179 and 180 and a 3′-primer        of SEQ ID NO: 51, for amplification of a first binding domain;        and/or    -   b. a mix of primers comprising a 5′-primer selected from the        group consisting of SEQ ID NOs: 121, 127, 139, 154, 155, and 175        and a 3′-primer selected from the group consisting of SEQ ID        NOs: 128, 129, 147, 176, and 177, for amplification of a second        binding domain.

In one embodiment (117), the invention relates to a monoclonal antibodyor a functional part thereof comprising a light chain (VL) and/or aheavy chain (VH) domain, which is encoded by a polynucleotide located ona nucleotide fragment that can be obtained by PCR amplification of DNAof hybridoma cell line A6-2G5-41 deposited on Aug. 30, 2011 as DSMACC3138 using

-   -   a. a mix of primers comprising a 5′-primer selected from the        group consisting of SEQ ID NOs: 51 and 188-192 and a 3′-primer        of SEQ ID NO: 51, for amplification of a first binding domain;        and/or    -   b. a mix of primers comprising a 5′-primer selected from the        group consisting of SEQ ID NOs: 120, 124, 126, 181, 182 and 183        and a 3′-primer selected from the group consisting of SEQ ID        NOs: 144, 145 and 184-187, for amplification of a second binding        domain.

In one embodiment (118), the invention relates to a monoclonal antibodyor a functional part thereof comprising a light chain (VL) and/or aheavy chain (VH) domain, which is encoded by a polynucleotide located ona nucleotide fragment that can be obtained by PCR amplification of DNAof hybridoma cell line A4-4A6-48 deposited on Aug. 30, 2011 as DSMACC3136 using

-   -   a. a mix of primers comprising a 5′-primer selected from the        group consisting of SEQ ID NOs: 50 and 201-204 and a 3′-primer        of SEQ ID NO: 51, for amplification of a first binding domain;        and/or    -   b. a mix of primers comprising a 5′-primer selected from the        group consisting of SEQ ID NOs: 121, 137, 151 and 193-197 and a        3′-primer selected from the group consisting of SEQ ID NOs: 131,        141, 144, 166, 198, 199 and 200, for amplification of a second        binding domain.

In one embodiment (119), the invention relates to a monoclonal antibodyor a functional part thereof comprising a light chain (VL) and/or aheavy chain (VH) domain, which is encoded by a polynucleotide located ona nucleotide fragment that can be obtained by PCR amplification of DNAof hybridoma cell line A6-1D2-12 deposited on Sep. 6, 2011 as DSMACC3141 using

-   -   a. a mix of primers comprising a 5′-primer selected from the        group consisting of SEQ ID NOs: 209-214, and 219-221 a 3′-primer        of SEQ ID NO: 215, for amplification of a first binding domain;        and/or    -   b. a mix of primers comprising a 5′-primer selected from the        group consisting of SEQ ID NOs: 216, 217 and 218 and a 3′-primer        of SEQ ID NOs: 208, for amplification of a second binding        domain.

In one embodiment (120), the antibody according to any one of thepreceding embodiments may be a polyclonal antibody, a monoclonalantibody, a chimeric antibody, a humanized antibody, a human antibody, acamelid antibody, a diabody, or a modified or engineered antibody.

In one embodiment (121), the binding peptide or functional part thereofmay be a fragment comprising a heavy chain and/or a light chain,particularly a heavy chain as show in SEQ ID NOs: 1-5 and/or a lightchain as shown in SEQ ID NOs: 6-11, particularly a Fab or a F(ab′)₂fragment.

In a specific embodiment (122), the invention relates to a heavy chainas show in SEQ ID NOs: 1-5.

In another specific embodiment (123), the invention relates to a lightchain as shown in SEQ ID NOs: 6-11.

In one embodiment (124), the invention provides a method for producingthe binding peptides or antibodies of any one of the precedingembodiments, comprising the step of culturing the cell line of any ofthe preceding embodiments in a suitable cultivation medium and,optionally, purifying the binding peptides or antibody from the cellline or cultivation medium.

BRIEF DESCRIPTION OF FIGURES AND SEQUENCES Figures

FIGS. 1A and 1B show antibody binding to phospho-Tau in brain sectionsfrom biGT (Tau bigenic) mice using TAUPIR.

FIG. 2 shows antibody binding to phospho-Tau in brain sections from ADand tauopathy patients using TAUPIR using ACI-36-3A8-Ab1 antibody.

FIG. 3 shows the effect of anti-Tau antibody treatment following 1 weekin vivo study on pTau epitope pT231 using MSD.

FIGS. 4A and 4B show a diagram demonstrating how brains were preparedfor soluble and sarkosyl insoluble (Sin T) Tau protein fractions.

FIGS. 5A through 51 show pTau epitope Western Blot results afteranti-Tau antibody treatment for the 1 month (FIG. 5A, 5B, 5C, 5G, 5H,5I) or 3 month in vivo study (FIG. 5D, 5E, 5F)

FIGS. 6A through 6O show pTau epitope Western Blot results afteranti-Tau antibody treatment for the 3 month in vivo study using biGTbigenic mice.

FIG. 7 shows IHC after anti-Tau antibody treatment by ACI-36-2B6-Ab1 in3 month in vivo study.

FIG. 8 shows IHC after anti-Tau antibody treatment by ACI-36-3A8-Ab1 in3 month in vivo study.

FIG. 9 shows the Morris Water-Maze results after anti-Tau antibodytreatment by ACI-36-2B6-Ab1 in 3 month in vivo study.

FIG. 10 shows the Morris Water-Maze results after anti-Tau antibodytreatment by ACI-36-3A8-Ab1 in 3 month in vivo study.

SEQUENCES

SEQ ID NO: 1 depicts the amino acid sequence of the heavy chain variableregion (VH) of monoclonal antibody ACI-36-3A8-Ab1 produced by hybridomacell line 3A8A12G7.

SEQ ID NO: 2 depicts the amino acid sequence of the heavy chain variableregion (VH) of monoclonal antibody ACI-36-2B6-Ab1 produced by hybridomacell line 2B6A10C11.

SEQ ID NO: 3 depicts the amino acid sequence of the heavy chain variableregion (VH) of monoclonal antibody ACI-36-6H1-Ab1 and ACI-36-6H1-Ab2produced by hybridoma cell line 6H1A11C11 and 6H1G6E6, respectively.

SEQ ID NO: 4 depicts the amino acid sequence of the heavy chain variableregion (VH) of monoclonal antibody ACI-33-6C10-Ab2 and ACI-33-6C10-Ab1produced by hybridoma cell line 6C10E5E9C12 and 6C10F9C12A11,respectively.

SEQ ID NO: 5 depicts the amino acid sequence of the heavy chain variableregion (VH) of monoclonal antibody ACI-41-7C2-Ab1 and ACI-41-7C2-Ab2produced by hybridoma cell line 7C2(1)F10C10D3 and 7C2(2)B9F11D5,respectively.

SEQ ID NO: 6 depicts the amino acid sequence of the light chain variableregion (VK) of monoclonal antibody ACI-36-3A8-Ab1_(VK-AD) andACI-36-3A8-Ab2_(VK-AD) produced by hybridoma cell line 3A8A12G7 and3A8E12H8, respectively.

SEQ ID NO: 7 depicts the amino acid sequence of the light chain variableregion (VK) of monoclonal antibody ACI-36-3A8-Ab1_(VK-G) andACI-36-3A8-Ab2_(VK-G) produced by hybridoma cell line 3A8A12G7 and3A8E12H8, respectively.

SEQ ID NO: 8 depicts the amino acid sequence of the light chain variableregion (VK) of monoclonal antibody ACI-36-2B6-Ab1 and ACI-36-2B6-Ab2produced by hybridoma cell line 2B6A10C11 and 2B6G7A12, respectively.

SEQ ID NO: 9 depicts the amino acid sequence of the light chain variableregion (VK) of monoclonal antibody ACI-36-6H1-Ab1 and ACI-36-6H1-Ab2produced by hybridoma cell line 6H1A11C11 and 6H1G6E6, respectively.

SEQ ID NO: 10 depicts the amino acid sequence of the light chainvariable region (VK) of monoclonal antibody ACI-33-6C10-Ab2 andACI-33-6C10-Ab1 produced by hybridoma cell line 6C10E5E9C12 and6C10F9C12A11, respectively.

SEQ ID NO: 11 depicts the amino acid sequence of the light chainvariable region (VK) of monoclonal antibody ACI-41-7C2-Ab1 andACI-41-7C2-Ab2 produced by hybridoma cell line 7C2(1)F10C10D3 and7C2(2)B9F11D5, respectively.

SEQ ID NO: 12 depicts the amino acid sequence of the CDR1 of the heavychain variable region (VH) of monoclonal antibody ACI-36-3A8-Ab1,ACI-36-3A8-Ab2, ACI-36-2B6-Ab1, ACI-36-2B6-Ab2, ACI-36-6H1-Ab1 andACI-36-6H1-Ab2 produced by hybridoma cell line 3A8A12G7, 3A8E12H8,2B6A10C11, 2B6G7A12, 6H1A11C11 and 6H1G6E6, respectively.

SEQ ID NO: 13 depicts the amino acid sequence of the CDR2 of the heavychain variable region (VH) of monoclonal antibody ACI-36-3A8-Ab1,ACI-36-3A8-Ab2, ACI-36-2B6-Ab1, ACI-36-2B6-Ab2, ACI-36-6H1-Ab1 andACI-36-6H1-Ab2 produced by hybridoma cell line 3A8A12G7, 3A8E12H8,2B6A10C11, 2B6G7A12, 6H1A11C11 and 6H1G6E6, respectively.

SEQ ID NO: 14 depicts the amino acid sequence of the CDR3 of the heavychain variable region (VH) of monoclonal antibody ACI-36-3A8-Ab1,ACI-36-3A8-Ab2, ACI-36-2B6-Ab1, ACI-36-2B6-Ab2, ACI-36-6H1-Ab1 andACI-36-6H1-Ab2 produced by hybridoma cell line 3A8A12G7, 3A8E12H8,2B6A10C11, 2B6G7A12, 6H1A11C11 and 6H1G6E6, respectively.

SEQ ID NO: 15 depicts the amino acid sequence of the CDR1 of the heavychain variable region (VH) of monoclonal antibody ACI-33-6C10-Ab2 andACI-33-6C10-Ab1 produced by hybridoma cell line 6C10E5E9C12 and6C10F9C12A11, respectively.

SEQ ID NO: 16 depicts the amino acid sequence of the CDR2 of the heavychain variable region (VH) of monoclonal antibody ACI-33-6C10-Ab2 andACI-33-6C10-Ab1 produced by hybridoma cell line 6C10E5E9C12 and6C10F9C12A11, respectively.

SEQ ID NO: 17 depicts the amino acid sequence of the CDR3 of the heavychain variable region (VH) of monoclonal antibody ACI-33-6C10-Ab2 andACI-33-6C10-Ab1 produced by hybridoma cell line 6C10E5E9C12 and6C10F9C12A11, respectively.

SEQ ID NO: 18 depicts the amino acid sequence of the CDR1 of the heavychain variable region (VH) of monoclonal antibody ACI-41-7C2-Ab1 andACI-41-7C2-Ab2 produced by hybridoma cell line 7C2(1)F10C10D3 and7C2(2)B9F11D5, respectively.

SEQ ID NO: 19 depicts the amino acid sequence of the CDR2 of the heavychain variable region (VH) of monoclonal antibody ACI-41-7C2-Ab1 andACI-41-7C2-Ab2 produced by hybridoma cell line 7C2(1)F10C10D3 and7C2(2)B9F11D5, respectively.

SEQ ID NO: 20 depicts the amino acid sequence of the CDR3 of the heavychain variable region (VH) of monoclonal antibody ACI-41-7C2-Ab1 andACI-41-7C2-Ab2 produced by hybridoma cell line 7C2(1)F10C10D3 and7C2(2)B9F11D5, respectively.

SEQ ID NO: 21 depicts the amino acid sequence of the CDR1 of the lightchain variable region (VK) of monoclonal antibody ACI-36-3A8-Ab1_(VK-AD)and ACI-36-3A8-Ab2_(VK-AD) produced by hybridoma cell line 3A8A12G7 and3A8E12H8, respectively.

SEQ ID NO: 22 depicts the amino acid sequence of the CDR2 of the lightchain variable region (VK) of monoclonal antibody ACI-36-3A8-Ab1_(VK-AD)and ACI-36-3A8-Ab2_(VK-AD) produced by hybridoma cell line 3A8A12G7 and3A8E12H8, respectively.

SEQ ID NO: 23 depicts the amino acid sequence of the CDR3 of the lightchain variable region (VK) of monoclonal antibody ACI-36-3A8-Ab1_(VK-AD)and ACI-36-3A8-Ab2_(VK-AD) produced by hybridoma cell line 3A8A12G7 and3A8E12H8, respectively.

SEQ ID NO: 24 depicts the amino acid sequence of the CDR1 of the lightchain variable region (VK) of monoclonal antibody ACI-36-3A8-Ab1_(VK-G)and ACI-36-3A8-Ab2_(VK-G) produced by hybridoma cell line 3A8A12G7 and3A8E12H8, respectively.

SEQ ID NO: 25 depicts the amino acid sequence of the CDR2 of the lightchain variable region (VK) of monoclonal antibody ACI-36-3A8-Ab1 VK-G,ACI-36-3A8-Ab2_(VK-G), ACI-36-2B6-Ab1, ACI-36-2B6-Ab2, ACI-36-6H1-Ab1and ACI-36-6H1-Ab2 produced by hybridoma cell line 3A8A12G7, 3A8E12H8,2B6A10C11, 2B6G7A12, 6H1A11C11 and 6H1G6E6, respectively.

SEQ ID NO: 26 depicts the amino acid sequence of the CDR3 of the lightchain variable region (VK) of monoclonal antibody ACI-36-3A8-Ab1 VK-G,ACI-36-3A8-Ab2_(VK-G), ACI-36-2B6-Ab1, ACI-36-2B6-Ab2, ACI-36-6H1-Ab1and ACI-36-6H1-Ab2 produced by hybridoma cell line 3A8A12G7, 3A8E12H8,2B6A10C11, 2B6G7A12, 6H1A11C11 and 6H1G6E6, respectively.

SEQ ID NO: 27 depicts the amino acid sequence of the CDR1 of the lightchain variable region (VK) of monoclonal antibody ACI-36-2B6-Ab1 andACI-36-2B6-Ab2 produced by hybridoma cell line 2B6A10C11 and 2B6G7A12,respectively.

SEQ ID NO: 28 depicts the amino acid sequence of the CDR1 of the lightchain variable region (VK) of monoclonal antibody ACI-36-6H1-Ab1 andACI-36-6H1-Ab2 produced by hybridoma cell line 6H1A11C11 and 6H1G6E6,respectively.

SEQ ID NO: 29 depicts the amino acid sequence of the CDR1 of the lightchain variable region (VK) of monoclonal antibody ACI-33-6C10-Ab2 andACI-33-6C10-Ab1 produced by hybridoma cell line 6C10E5E9C12 and6C10F9C12A11, respectively.

SEQ ID NO: 30 depicts the amino acid sequence of the CDR2 of the lightchain variable region (VK) of monoclonal antibody ACI-33-6C10-Ab2 andACI-33-6C10-Ab1 produced by hybridoma cell line 6C10E5E9C12 and6C10F9C12A11, respectively.

SEQ ID NO: 31 depicts the amino acid sequence of the CDR3 of the lightchain variable region (VK) of monoclonal antibody ACI-33-6C10-Ab2 andACI-33-6C10-Ab1 produced by hybridoma cell line 6C10E5E9C12 and6C10F9C12A11, respectively.

SEQ ID NO: 32 depicts the amino acid sequence of the CDR1 of the lightchain variable region (VK) of monoclonal antibody ACI-41-7C2-Ab1 andACI-41-7C2-Ab2 produced by hybridoma cell line 7C2(1)F10C10D3 and7C2(2)B9F11D5, respectively.

SEQ ID NO: 33 depicts the amino acid sequence of the CDR2 of the lightchain variable region (VK) of monoclonal antibody ACI-41-7C2-Ab1 andACI-41-7C2-Ab2 produced by hybridoma cell line 7C2(1)F10C10D3 and7C2(2)B9F11D5, respectively.

SEQ ID NO: 34 depicts the amino acid sequence of the CDR3 of the lightchain variable region (VK) of monoclonal antibody ACI-41-7C2-Ab1 andACI-41-7C2-Ab2 produced by hybridoma cell line 7C2(1)F10C10D3 and7C2(2)B9F11D5, respectively.

SEQ ID NO: 35 depicts the nucleotide sequence of the heavy chainvariable region (VH) of monoclonal antibody ACI-36-3A8-Ab1 andACI-36-3A8-Ab2 produced by hybridoma cell line 3A8A12G7 and 3A8E12H8,respectively.

SEQ ID NO: 36 depicts the nucleotide sequence of the heavy chainvariable region (VH) of monoclonal antibody ACI-36-2B6-Ab1 andACI-36-2B6-Ab2 produced by hybridoma cell line 2B6A10C11 and 2B6G7A12,respectively.

SEQ ID NO: 37 depicts the nucleotide sequence of the heavy chainvariable region (VH) of monoclonal antibody ACI-36-6H1-Ab1 andACI-36-6H1-Ab2 produced by hybridoma cell line 6H1A11C11 and 6H1G6E6,respectively.

SEQ ID NO: 38 depicts the nucleotide sequence of the heavy chainvariable region (VH) of monoclonal antibody ACI-33-6C10-Ab2 andACI-33-6C10-Ab1 produced by hybridoma cell line 6C10E5E9C12 and6C10F9C12A11, respectively.

SEQ ID NO: 39 depicts the nucleotide sequence of the heavy chainvariable region (VH) of monoclonal antibody ACI-41-7C2-Ab1 andACI-41-7C2-Ab2 produced by hybridoma cell line 7C2(1)F10C10D3 and7C2(2)B9F11D5, respectively.

SEQ ID NO: 40 depicts the nucleotide sequence of the light chainvariable region (VK) of monoclonal antibody ACI-36-3A8-Ab1 andACI-36-3A8-Ab2 produced by hybridoma cell line 3A8A12G7 and 3A8E12H8,respectively.

SEQ ID NO: 41 depicts the nucleotide sequence of the light chainvariable region (VK) of monoclonal antibody ACI-36-3A8-Ab1 andACI-36-3A8-Ab2 produced by hybridoma cell line 3A8A12G7 and 3A8E12H8,respectively.

SEQ ID NO: 42 depicts the nucleotide sequence of the light chainvariable region (VK) of monoclonal antibody ACI-36-2B6-Ab1 andACI-36-2B6-Ab2 produced by hybridoma cell line 2B6A10C11 and 2B6G7A12,respectively.

SEQ ID NO: 43 depicts the nucleotide sequence of the light chainvariable region (VK) of monoclonal antibody ACI-36-6H1-Ab1 andACI-36-6H1-Ab2 produced by hybridoma cell line 6H1A11C11 and 6H1G6E6,respectively.

SEQ ID NO: 44 depicts the nucleotide sequence of the light chainvariable region (VK) of monoclonal antibody ACI-33-6C10-Ab2 andACI-33-6C10-Ab1 produced by hybridoma cell line 6C10E5E9C12 and6C10F9C12A11, respectively.

SEQ ID NO: 45 depicts the nucleotide sequence of the light chainvariable region (VK) of monoclonal antibody ACI-41-7C2-Ab1 andACI-41-7C2-Ab2 produced by hybridoma cell line 7C2(1)F10C10D3 and7C2(2)B9F11D5, respectively.

SEQ ID NO: 46-57 depicts the nucleotide sequences of VH/VK forward andreverse primers.

SEQ ID NO: 58 depicts the amino acid sequence of Tau 379-408 [pS396,pS404]

SEQ ID NO: 59 depicts the amino acid sequence of Tau 5-20 [pY18]

SEQ ID NO: 60 depicts the amino acid sequence of Tau 206-221 [pT212,pS214]

SEQ ID NO: 61 depicts the amino acid sequence of Tau 196-211 [pS202,pT205]

SEQ ID NO: 62 depicts the amino acid sequence of Tau 393-408 [pS396,pS404]

SEQ ID NO: 63 depicts the amino acid sequence of Tau 401-418 [pS404,pS409]

SEQ ID NO: 64 depicts the amino acid sequence of Tau 200-216[pS202+pT205 & pT212+pS214]

SEQ ID NO: 65 depicts the amino acid sequence of Tau 407-418 [pS409]

SEQ ID NO: 66 depicts the amino acid sequence of Tau 399-408 [pS404]

SEQ ID NO: 67 depicts the amino acid sequence of longest isoform ofhuman Tau (441aa), also called Tau40

SEQ ID NO: 68 depicts the amino acid sequence of the heavy chainvariable region (VH) of monoclonal antibody ACI-35-4A6-Ab1 produced byhybridoma cell line A4-4A6-18.

SEQ ID NO: 69 depicts the amino acid sequence of the light chainvariable region (VK) of monoclonal antibody ACI-35-4A6-Ab1 produced byhybridoma cell line A4-4A6-18.

SEQ ID NO: 70 depicts the amino acid sequence of the CDR1 of the heavychain variable region (VH) of monoclonal antibody ACI-35-4A6-Ab1

SEQ ID NO: 71 depicts the amino acid sequence of the CDR2 of the heavychain variable region (VH) of monoclonal antibody ACI-35-4A6-Ab1

SEQ ID NO: 72 depicts the amino acid sequence of the CDR3 of the heavychain variable region (VH) of monoclonal antibody ACI-35-4A6-Ab1

SEQ ID NO: 73 depicts the amino acid sequence of the CDR1 of the lightchain variable region (VK) of monoclonal antibody ACI-35-4A6-Ab1

SEQ ID NO: 74 depicts the amino acid sequence of the CDR2 of the lightchain variable region (VK) of monoclonal antibody ACI-35-4A6-Ab1

SEQ ID NO: 75 depicts the amino acid sequence of the CDR3 of the lightchain variable region (VK) of monoclonal antibody ACI-35-4A6-Ab1

SEQ ID NO: 76 depicts the amino acid sequence of the heavy chainvariable region (VH) of monoclonal antibody ACI-35-1D2-Ab1 produced byhybridoma cell line A6-1D2-12.

SEQ ID NO: 77 depicts the amino acid sequence of the light chainvariable region (VK) of monoclonal antibody ACI-35-1D2-Ab1 produced byhybridoma cell line A6-1D2-12.

SEQ ID NO: 78 depicts the amino acid sequence of the CDR1 of the heavychain variable region (VH) of monoclonal antibody ACI-35-1 D2-Ab1.

SEQ ID NO: 79 depicts the amino acid sequence of the CDR2 of the heavychain variable region (VH) of monoclonal antibody ACI-35-1 D2-Ab1.

SEQ ID NO: 80 depicts the amino acid sequence of the CDR3 of the heavychain variable region (VH) of monoclonal antibody ACI-35-1 D2-Ab1

SEQ ID NO: 81 depicts the amino acid sequence of the CDR1 of the lightchain variable region (VK) of monoclonal antibody ACI-35-1D2-Ab1

SEQ ID NO: 82 depicts the amino acid sequence of the CDR2 of the lightchain variable region (VK) of monoclonal antibody ACI-35-1D2-Ab1

SEQ ID NO: 83 depicts the amino acid sequence of the CDR3 of the lightchain variable region (VK) of monoclonal antibody ACI-35-1D2-Ab1

SEQ ID NO: 84 depicts the nucleotide sequence of the heavy chainvariable region (VH) of monoclonal antibody ACI-35-4A6-Ab1 produced byhybridoma cell line A4-4A6-18.

SEQ ID NO: 85 depicts the nucleotide sequence of the light chainvariable region (VK) of monoclonal antibody ACI-35-4A6-Ab1 produced byhybridoma cell line A4-4A6-18.

SEQ ID NO: 86 depicts the nucleotide sequence of the heavy chainvariable region (VH) of monoclonal antibody ACI-35-1D2-Ab1 produced byhybridoma cell line A6-1D2-12.

SEQ ID NO: 87 depicts the nucleotide sequence of the light chainvariable region (VK) of monoclonal antibody ACI-35-1D2-Ab1 produced byhybridoma cell line A6-1D2-12.

SEQ ID NO: 88 depicts the amino acid sequence of the heavy chainvariable region (VH) of monoclonal antibody ACI-35-2A1-Ab1,ACI-35-2A1-Ab2, and ACI-35-4A6-Ab2, respectively, produced by hybridomacell line A4-2A1-18, A4-2A1-40 and A4-4A6-48, respectively.

SEQ ID NO: 89 depicts the amino acid sequence of the CDR1 of the heavychain variable region (VH) of monoclonal antibody ACI-35-2A1-Ab1,ACI-35-2A1-Ab2, ACI-35-4A6-Ab2, ACI-35-2G5-AB2 and ACI-35-2G5-AB3,respectively.

SEQ ID NO: 90 depicts the amino acid sequence of the CDR2 of the heavychain variable region (VH) of monoclonal antibody ACI-35-2A1-Ab1,ACI-35-2A1-Ab2, and ACI-35-4A6-Ab2, respectively.

SEQ ID NO: 91 depicts the amino acid sequence of the CDR3 of the heavychain variable region (VH) of monoclonal antibody ACI-35-2A1-Ab1,ACI-35-2A1-Ab2, ACI-35-4A6-Ab2, ACI-35-2G5-AB2 and ACI-35-2G5-AB3,respectively.

SEQ ID NO: 92 depicts the amino acid sequence of the light chainvariable region (VK) of monoclonal antibody ACI-35-2A1-Ab2 produced byhybridoma cell line A4-2A1-40

SEQ ID NO: 93 depicts the amino acid sequence of the CDR1 of the lightchain variable region (VK) of monoclonal antibody ACI-35-2A1-Ab2.

SEQ ID NO: 94 depicts the amino acid sequence of the CDR2 of the lightchain variable region (VK) of monoclonal antibody ACI-35-2A1-Ab2.

SEQ ID NO: 95 depicts the amino acid sequence of the CDR3 of the lightchain variable region (VK) of monoclonal antibody ACI-35-2A1-Ab2.

SEQ ID NO: 96 depicts the amino acid sequence of the heavy chainvariable region (VH) of monoclonal antibody ACI-35-2G5-Ab1 produced byhybridoma cell line A6-2G5-08.

SEQ ID NO: 97 depicts the amino acid sequence of the light chainvariable region (VK) of monoclonal antibody ACI-35-2G5-Ab1 produced byhybridoma cell line A6-2G5-08.

SEQ ID NO: 98 depicts the amino acid sequence of the CDR1 of the heavychain variable region (VH) of monoclonal antibody ACI-35-2G5-Ab1.

SEQ ID NO: 99 depicts the amino acid sequence of the CDR2 of the heavychain variable region (VH) of monoclonal antibody ACI-35-2G5-Ab1.

SEQ ID NO: 100 depicts the amino acid sequence of the CDR3 of the heavychain variable region (VH) of monoclonal antibody ACI-35-2G5-Ab1.

SEQ ID NO: 101 depicts the amino acid sequence of the CDR1 of the lightchain variable region (VK) of monoclonal antibody ACI-35-2G5-Ab1.

SEQ ID NO: 102 depicts the amino acid sequence of the CDR2 of the lightchain variable region (VK) of monoclonal antibody ACI-35-2G5-Ab1.

SEQ ID NO: 103 depicts the amino acid sequence of the CDR3 of the lightchain variable region (VK) of monoclonal antibody ACI-35-2G5-Ab1.

SEQ ID NO: 104 depicts the amino acid sequence of the heavy chainvariable region (VH) of monoclonal antibody ACI-35-2G5-AB2 andACI-35-2G5-AB3, respectively, produced by hybridoma cell line A6-2G5-30and A6-2G5-41, respectively.

SEQ ID NO: 105 depicts the amino acid sequence of the light chainvariable region (VK) of monoclonal antibody ACI-35-2G5-AB2 andACI-35-2G5-AB3, respectively, produced by hybridoma cell line A6-2G5-30and A6-2G5-41, respectively.

SEQ ID NO: 106 depicts the amino acid sequence of the CDR1 of the lightchain variable region (VK) of monoclonal antibody ACI-35-2G5-AB2 andACI-35-2G5-AB3, respectively.

SEQ ID NO: 107 depicts the amino acid sequence of the CDR2 of the lightchain variable region (VK) of monoclonal antibody ACI-35-2G5-AB2 andACI-35-2G5-AB3, respectively.

SEQ ID NO: 108 depicts the amino acid sequence of the CDR3 of the lightchain variable region (VK) of monoclonal antibody ACI-35-2G5-AB2 andACI-35-2G5-AB3, respectively.

SEQ ID NO: 109 depicts the nucleotide sequence of the heavy chainvariable region (VH) of monoclonal antibody ACI-35-2A1-Ab1,ACI-35-2A1-Ab2, and ACI-35-4A6-Ab2, respectively, produced by hybridomacell line A4-2A1-18, A4-2A1-40 and A4-4A6-48, respectively.

SEQ ID NO: 110 depicts the nucleotide sequence of the light chainvariable region (VK) of monoclonal antibody ACI-35-2A1-Ab2 produced byhybridoma cell line A4-2A1-40.

SEQ ID NO: 111 depicts the nucleotide sequence of the heavy chainvariable region (VH) of monoclonal antibody ACI-35-2G5-AB1 produced byhybridoma cell line A6-2G5-08.

SEQ ID NO: 112 depicts the nucleotide sequence of the light chainvariable region (VK) of monoclonal antibody ACI-35-2G5-AB1 produced byhybridoma cell line A6-2G5-08.

SEQ ID NO: 113 depicts the nucleotide sequence of the heavy chainvariable region (VH) of monoclonal antibody ACI-35-2G5-AB2 andACI-35-2G5-AB3, respectively, produced by hybridoma cell line A6-2G5-30and A6-2G5-41, respectively.

SEQ ID NO: 114 depicts the nucleotide sequence of the light chainvariable region (VK) of monoclonal antibody ACI-35-2G5-AB2 andACI-35-2G5-AB3, respectively, produced by hybridoma cell line A6-2G5-30and A6-2G5-41, respectively.

SEQ ID NO: 115 depicts the amino acid sequence of the CDR2 of the heavychain variable region (VH) of monoclonal antibody ACI-35-2G5-AB2 andACI-35-2G5-AB3.

SEQ ID NO: 116 depicts the amino acid sequence of the light chainvariable region (VK) of monoclonal antibody ACI-35-2A1-Ab1 produced byhybridoma cell line A4-2A1-18.

SEQ ID NO: 117 depicts the nucleotide sequence of the light chainvariable region (VK) of monoclonal antibody ACI-35-2A1-Ab1 produced byhybridoma cell line A4-2A1-18.

SEQ ID NO: 118 depicts the amino acid sequence of the light chainvariable region (VK) of monoclonal antibody ACI-35-4A6-Ab2 produced byhybridoma cell line A4-4A6-48.

SEQ ID NO: 119 depicts the nucleotide sequence of the light chainvariable region (VK) of monoclonal antibody ACI-35-4A6-Ab2 produced byhybridoma cell line A4-4A6-48.

SEQ ID NO: 120-221 depicts the nucleotide sequences of VH/VK forward andreverse primers

DEFINITION OF TERMS

The terms “polypeptide”, “peptide”, and “protein”, as used herein, areinterchangeably and are defined to mean a biomolecule composed of aminoacids linked by a peptide bond.

The term “peptides,” or “binding peptide” are used hereininterchangeably and refer to chains of amino acids (typically L-aminoacids) whose alpha carbons are linked through peptide bonds formed by acondensation reaction between the carboxyl group of the alpha carbon ofone amino acid and the amino group of the alpha carbon of another aminoacid. The terminal amino acid at one end of the chain (i.e., the aminoterminal) has a free amino group, while the terminal amino acid at theother end of the chain (i.e., the carboxy terminal) has a free carboxylgroup. As such, the term “amino terminus” (abbreviated N-terminus)refers to the free alpha-amino group on the amino acid at the aminoterminal of the peptide, or to the alpha-amino group (imino group whenparticipating in a peptide bond) of an amino acid at any other locationwithin the peptide. Similarly, the term “carboxy terminus” (abbreviatedC-terminus) refers to the free carboxyl group on the amino acid at thecarboxy terminus of a peptide, or to the carboxyl group of an amino acidat any other location within the peptide. A binding peptide mayconstitutes antibodies such as polyclonal or monoclonal antibodies,human or humanized antibodies, diabodies, camelid antibodies, etc, orfunctional parts thereof as defined herein.

The terms “fragment thereof” or “fragment” as used herein refer to afunctional peptide fragment which has essentially the same (biological)activity as the peptides defined herein (e.g. as shown in SEQ ID NOs59-66 in Table 1 respectively), i.e. said fragments are still capable ofeliciting a highly specific, particularly a conformation specific,immune response in an organism, but particularly within an animal,particularly a mammal or a human, which is highly effective and capableof preventing or alleviating tauopathies, or the symptoms associatedwith tauopathies. In particular, said fragments still contain thespecific pathological phospho-epitope or -epitopes of the tau peptide,as used and defined herein.

Typically, the amino acids making up a peptide are numbered in order,starting at the amino terminal and increasing in the direction towardthe carboxy terminal of the peptide. Thus, when one amino acid is saidto “follow” another, that amino acid is positioned closer to the carboxyterminal of the peptide than the preceding amino acid.

The term “residue” is used herein to refer to an amino acid that isincorporated into a peptide by an amide bond. As such, the amino acidmay be a naturally occurring amino acid or, unless otherwise limited,may encompass known analogs of natural amino acids that function in amanner similar to the naturally occurring amino acids (i.e., amino acidmimetics). Moreover, an amide bond mimetic includes peptide backbonemodifications well known to those skilled in the art.

The phrase “consisting essentially of” is used herein to exclude anyelements that would substantially alter the essential properties of thepeptides to which the phrase refers. Thus, the description of a peptide“consisting essentially of . . . ” excludes any amino acidsubstitutions, additions, or deletions that would substantially alterthe biological activity of that peptide.

Furthermore, one of skill will recognize that, as mentioned above,individual substitutions, deletions or additions which alter, add ordelete a single amino acid or a small percentage of amino acids(typically less than 5%, more typically less than 1%) in an encodedsequence are conservatively modified variations where the alterationsresult in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following six groupseach contain amino acids that are conservative substitutions for oneanother:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The phrases “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany it as found in its native state. Thus, the peptides describedherein do not contain materials normally associated with their in situenvironment. Typically, the isolated, immunogenic peptides describedherein are at least about 80% pure, usually at least about 90%, andpreferably at least about 95% as measured by band intensity on a silverstained gel.

Protein purity or homogeneity may be indicated by a number of methodswell known in the art, such as polyacrylamide gel electrophoresis of aprotein sample, followed by visualization upon staining. For certainpurposes high resolution will be needed and HPLC or a similar means forpurification utilized.

When the immunogenic peptides are relatively short in length (i.e., lessthan about 50 amino acids), they are often synthesized using standardchemical peptide synthesis techniques.

Solid phase synthesis in which the C-terminal amino acid of the sequenceis attached to an insoluble support followed by sequential addition ofthe remaining amino acids in the sequence is a preferred method for thechemical synthesis of the immunogenic peptides described herein.Techniques for solid phase synthesis are known to those skilled in theart.

Alternatively, the immunogenic peptides described herein are synthesizedusing recombinant nucleic acid methodology. Generally, this involvescreating a nucleic acid sequence that encodes the peptide, placing thenucleic acid in an expression cassette under the control of a particularpromoter, expressing the peptide in a host, isolating the expressedpeptide or polypeptide and, if required, renaturing the peptide.Techniques sufficient to guide one of skill through such procedures arefound in the literature.

Once expressed, recombinant peptides can be purified according tostandard procedures, including ammonium sulfate precipitation, affinitycolumns, column chromatography, gel electrophoresis and the like.Substantially pure compositions of about 50% to 95 homogeneity arepreferred, and 80% to 95% or greater homogeneity is most preferred foruse as therapeutic agents.

One of skill in the art will recognize that after chemical synthesis,biological expression or purification, the immunogenic peptides maypossess a conformation substantially different than the nativeconformations of the constituent peptides. In this case, it is oftennecessary to denature and reduce the antiproliferative peptide and thento cause the peptide to re-fold into the preferred conformation. Methodsof reducing and denaturing proteins and inducing re-folding are wellknown to those of skill in the art.

Antigenicity of the purified protein may be confirmed, for example, bydemonstrating reaction with immune serum, or with antisera producedagainst the protein itself.

The terms “a”, an and “the” as used herein are defined to mean “one ormore” and include the plural unless the context is inappropriate.

The terms “detecting” or “detected” as used herein mean using knowntechniques for detection of biologic molecules such as immunochemical orhistological methods and refer to qualitatively or quantitativelydetermining the presence or concentration of the biomolecule underinvestigation.

By “isolated” is meant a biological molecule free from at least some ofthe components with which it naturally occurs.

The terms “antibody”, “antibodies” or “functional parts thereof” as usedherein is an art recognized term and is understood to refer to moleculesor active fragments of molecules that bind to known antigens,particularly to immunoglobulin molecules and to immunologically activeportions of immunoglobulin molecules, i.e molecules that contain abinding site that immunospecifically binds an antigen. Theimmunoglobulin according to the invention can be of any type (IgG, IgM,IgD, IgE, IgA and IgY) or class (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2)or subclasses of immunoglobulin molecule.

“Antibodies” are intended within the scope of the present invention toinclude monoclonal antibodies, polyclonal, chimeric, single chain,bispecific, simianized, human and humanized antibodies, camelidantibodies, diabodies, as well as functional parts or active fragmentsthereof. Examples of active fragments of molecules that bind to knownantigens include Fab and F(ab′)₂ fragments, including the products of aFab immunoglobulin expression library and epitope-binding fragments ofany of the antibodies and fragments mentioned above. These activefragments can be derived from an antibody of the present invention by anumber of techniques. For example, purified monoclonal antibodies can becleaved with an enzyme, such as pepsin, and subjected to HPLC gelfiltration. The appropriate fraction containing Fab fragments can thenbe collected and concentrated by membrane filtration and the like. Forfurther description of general techniques for the isolation of activefragments of antibodies, see for example, Khaw, B. A. et al. J. Nucl.Med. 23:1011-1019 (1982); Rousseaux et al. Methods Enzymology,121:663-69, Academic Press, (1986).

A “humanized antibody” refers to a type of engineered antibody havingits CDRs derived from a non-human donor immunoglobulin, the remainingimmunoglobulin-derived parts of the molecule being derived from one (ormore) human immunoglobulin(s).

A humanized antibody may further refer to an antibody having a variableregion where one or more of its framework regions have human or primateamino acids. In addition, framework support residues may be altered topreserve binding affinity. Methods to obtain “humanized antibodies” arewell known to those skilled in the art. (see, e.g., Queen et al., Proc.Natl Acad Sci USA, 86:10029-10032 (1989), Hodgson et al., Bio/Technoloy,9:421 (1991)).

A “humanized antibody” may also be obtained by a novel geneticengineering approach that enables production of affinity-maturedhumanlike polyclonal antibodies in large animals such as, for example,rabbits (http://www.rctech.com/bioventures/therapeutic.php).

The term “fully human antibody” or “human” antibody is meant to refer toan antibody derived from transgenic mice carrying human antibody genesor from human cells. To the human immune system, however, the differencebetween “fully human”, “human”, and “humanized” antibodies may benegligible or nonexistent and as such all three may be of equal efficacyand safety.

The term “monoclonal antibody” is also well recognized in the art andrefers to an antibody that is mass produced in the laboratory from asingle clone and that recognizes only one antigen. Monoclonal antibodiesare typically made by fusing a normally short-lived, antibody-producingB cell to a fast-growing cell, such as a cancer cell (sometimes referredto as an “immortal” cell). The resulting hybrid cell, or hybridoma,multiplies rapidly, creating a clone that produces large quantities ofthe antibody.

The term “antigen” refers to an entity or fragment thereof which caninduce an immune response in an organism, particularly an animal, moreparticularly a mammal including a human. The term includes immunogensand regions responsible for antigenicity or antigenic determinants.

As used herein, the term “soluble” means partially or completelydissolved in an aqueous solution.

Also as used herein, the term “immunogenic” refers to substances whichelicit or enhance the production of antibodies, T-cells and otherreactive immune cells directed against an immunogenic agent andcontribute to an immune response in humans or animals.

An immune response occurs when an individual produces sufficientantibodies, T-cells and other reactive immune cells against administeredimmunogenic compositions of the present invention to moderate oralleviate the disorder to be treated.

The term “hybridoma” is art recognized and is understood by those ofordinary skill in the art to refer to a cell produced by the fusion ofan antibody-producing cell and an immortal cell, e.g. a multiple myelomacell. This hybrid cell is capable of producing a continuous supply ofantibody. See the definition of “monoclonal antibody” above and theExamples below for a more detailed description of the method of fusion.

The term “carrier” as used herein means a structure in which antigenicpeptide or supramolecular construct can be incorporated into or can beassociated with, thereby presenting or exposing antigenic peptides orpart of the peptide to the immune system of a human or animal. Anyparticle that can be suitably used in animal or human therapy such as,for example, a vesicle, a particle or a particulate body may be used asa carrier within the context of the present invention.

The term “carrier” further comprises methods of delivery whereinsupramolecular antigenic construct compositions comprising the antigenicpeptide may be transported to desired sites by delivery mechanisms. Oneexample of such a delivery system utilizes colloidal metals such ascolloidal gold.

Carrier proteins that can be used in the supramolecular antigenicconstruct compositions of the present invention include, but are notlimited to, maltose binding peptide “MBP”; bovine serum albumin “BSA”;keyhole lympet hemocyanin “KLH”; ovalbumin; flagellin; thyroglobulin;serum albumin of any species; gamma globulin of any species; syngeneiccells; syngeneic cells bearing Ia antigens; and polymers of D- and/orL-amino acids.

Further, the term “therapeutically effective amount” or“pharmaceutically effective amount” refers to the amount of bindingpeptide which, when administered to a human or animal, is sufficient toresult in a therapeutic effect in said human or animal. The effectiveamount is readily determined by one of ordinary skill in the artfollowing routine procedures.

“pTau PHF”, “PHF”, and “paired helical filaments” are used hereinsynonymously and refer to pairs of approximately 10 nm filaments woundinto helices with a periodicity of 160 nm visible on electronmicroscopy. Width varies between 10 and 22 nm. PHF are the predominantstructures in neurofibrillary tangles of Alzheimer's Disease (AD) andneuropil threads. PHF may also be seen in some but not all dystrophicneurites associated with neuritic plaques. The major component of PHF isa hyperphosphorylated form of microtubule-associated protein tau. PHFare composed of disulfide-linked antiparallel hyper-phosphorylated tauproteins. PHF tau may be truncated of its C-terminal 20 amino acidresidues. The mechanisms underlying PHF formation are uncertain buthyper-phosphorylation of tau may disengage it from microtubules,increasing the soluble pool of tau.

Within the scope of the present invention, it was demonstrated that theantibody induced response to the antigenic composition according to theinvention is largely T-cell independent. A nude mouse model was used inthis respect and nude mice were vaccinated and antibody responsesmeasured to evaluate the Aβ-specific antibody response induced by theantigenic composition according to the invention in the immunized nudemice. The nude mice carry the Foxn1nu mutation and as a consequence,have reduced T-cell function due to the lack of a proper thymus.

A “pharmaceutically effective amount” as used herein refers to a dose ofthe active ingredient in a pharmaceutical composition adequate to cure,or at least partially arrest, the symptoms of the disease, disorder orcondition to be treated or any complications associated therewith.

The present invention provides binding peptides recognizing and bindingto major pathological phospho-epitopes of the tau protein. Inparticular, the present invention provides specific antibodies againstlinear and conformational, simple and complex phospho-epitopes onprotein tau that are believed to be responsible for synapto- andneuro-toxicity in tauopathies, including AD.

Accordingly, the present invention relates in one embodiment to abinding peptide or a functional part thereof, particularly to anantibody, particularly a monoclonal antibody or a functional partthereof, which binding peptide or antibody recognizes and specificallybinds to a phospho-epitope on a mammalian, particularly on the human Tauprotein or on a fragment thereof, particularly to a pathological proteintau conformer, but, in one embodiment, does not bind to thecorresponding unphosphorylated epitope and/or to non-related epitopes,wherein said binding peptide or antibody has a high binding affinitywith a dissociation constant of at least 10 nM, particularly of at least8 nM, particularly of at least 5 nM, particularly of at least 2 nM,particularly of at least 1 nM, particularly of at least 500 pM,particularly of at least 400 pM particularly of at least 300 pM,particularly of at least 200 pM, particularly of at least 100 pM,particularly of at least 50 pM.

“Soluble Tau” protein as used herein refers to proteins consisting ofboth completely solubilized Tau protein/peptide monomers or of Tau-likepeptides/proteins, or of modified or truncated Tau peptides/proteins orof other derivates of Tau peptides/proteins monomers, and of Tau proteinoligomers. “Soluble Tau” excludes particularly neurofibrillary tangles(NFT).

“Insoluble Tau” as used herein refers to multiple aggregated monomers ofTau peptides or proteins, or of Tau-like peptides/proteins, or ofmodified or truncated Tau peptides/proteins or of other derivates of Taupeptides/proteins forming oligomeric or polymeric structures which areinsoluble both in vitro in aqueous medium and in vivo in the mammalianor human body more particularly in the brain, but particularly tomultiple aggregated monomers of Tau or of modified or truncated Taupeptides/proteins or of derivatives thereof, which are insoluble in themammalian or human body more particularly in the brain, respectively.“Insoluble Tau” particularly includes neurofibrillary tangles (NFT).

“Monomeric Tau” or “Tau monomer” as used herein refers to completelysolubilized Tau proteins without aggregated complexes in aqueous medium.

“Aggregated Tau”, “oligomeric Tau” and “Tau oligomer” refer to multipleaggregated monomers of Tau peptides or proteins, or of Tau-likepeptides/proteins, or of modified or truncated Tau peptides/proteins orof other derivates of Tau peptides/proteins forming oligomeric orpolymeric structures which are insoluble or soluble both in vitro inaqueous medium and in vivo in the mammalian or human body moreparticularly in the brain, but particularly to multiple aggregatedmonomers of Tau or of modified or truncated Tau peptides/proteins or ofderivatives thereof, which are insoluble or soluble in the mammalian orhuman body more particularly in the brain, respectively.”

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising a binding peptide or a functional part thereof,particularly an antibody, particularly a monoclonal antibody or afunctional part thereof, or a polynucleotide comprising a nucleic acidsequence encoding said binding peptide or antibody, according to any oneof the embodiments described and claimed herein, or a combinationthereof, in a therapeutically effective amount together with apharmaceutically acceptable carrier.

Suitable pharmaceutical carriers, diluents and/or excipients are wellknown in the art and include, for example, phosphate buffered salinesolutions, water, emulsions such as oil/water emulsions, various typesof wetting agents, sterile solutions, etc.

The binding peptides according to the invention including antibodies,particularly monoclonal antibodies and active fragments thereof, can beprepared in a physiologically acceptable formulation and may comprise apharmaceutically acceptable carrier, diluent and/or excipient usingknown techniques. For example, the binding peptides according to theinvention and as described herein including any functionally equivalentbinding peptides or functional parts thereof, in particular, themonoclonal antibodies of the invention including any functionallyequivalent antibodies or functional parts thereof, are combined with apharmaceutically acceptable carrier, diluent and/or excipient to form atherapeutic composition. Suitable pharmaceutical carriers, diluentsand/or excipients are well known in the art and include, for example,phosphate buffered saline solutions, water, emulsions such as oil/wateremulsions, various types of wetting agents, sterile solutions, etc.

Formulation of the pharmaceutical composition according to the inventioncan be accomplished according to standard methodology know to those ofordinary skill in the art.

The compositions of the present invention may be administered to asubject in the form of a solid, liquid or aerosol at a suitable,pharmaceutically effective dose. Examples of solid compositions includepills, creams, and implantable dosage units. Pills may be administeredorally. Therapeutic creams may be administered topically. Implantabledosage units may be administered locally, for example, at a tumor site,or may be implanted for systematic release of the therapeuticcomposition, for example, subcutaneously. Examples of liquidcompositions include formulations adapted for injection intramuscularly,subcutaneously, intravenously, intra-arterially, and formulations fortopical and intraocular administration. Examples of aerosol formulationsinclude inhaler formulations for administration to the lungs.

The compositions may be administered by standard routes ofadministration. In general, the composition may be administered bytopical, oral, rectal, nasal, interdermal, intraperitoneal, orparenteral (for example, intravenous, subcutaneous, or intramuscular)routes.

In addition, the composition may be incorporated into sustained releasematrices such as biodegradable polymers, the polymers being implanted inthe vicinity of where delivery is desired, for example, at the site of atumor. The method includes administration of a single dose,administration of repeated doses at predetermined time intervals, andsustained administration for a predetermined period of time.

A sustained release matrix, as used herein, is a matrix made ofmaterials, usually polymers which are degradable by enzymatic oracid/base hydrolysis or by dissolution. Once inserted into the body, thematrix is acted upon by enzymes and body fluids. The sustained releasematrix desirably is chosen by biocompatible materials such as liposomes,polylactides (polylactide acid), polyglycolide (polymer of glycolicacid), polylactide co-glycolide (copolymers of lactic acid and glycolicacid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid,collagen, chondroitin sulfate, carboxylic acids, fatty acids,phospholipids, polysaccharides, nucleic acids, polyamino acids, aminoacids such phenylalanine, tyrosine, isoleucine, polynucleotides,polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferredbiodegradable matrix is a matrix of one of either polylactide,polyglycolide, or polylactide co-glycolide (co-polymers of lactic acidand glycolic acid).

It is well known to those of ordinary skill in the pertinent art thatthe dosage of the composition will depend on various factors such as,for example, the condition of being treated, the particular compositionused, and other clinical factors such as weight, size, sex and generalhealth condition of the patient, body surface area, the particularcompound or composition to be administered, other drugs beingadministered concurrently, and the route of administration.

The composition according to the invention may be administered incombination with other compositions comprising an biologically activesubstance or compound such as, for example, a known compound used in themedication of tauopathies and/or of amyloidoses, a group of diseases anddisorders associated with amyloid or amyloid-like protein such as theamyloid β protein involved in Alzheimer's Disease.

The other biologically active substance or compound may exert itsbiological effect by the same or a similar mechanism as the therapeuticvaccine according to the invention or by an unrelated mechanism ofaction or by a multiplicity of related and/or unrelated mechanisms ofaction.

Generally, the other biologically active compound may includeneutron-transmission enhancers, psychotherapeutic drugs, acetylcholineesterase inhibitors, calcium-channel blockers, biogenic amines,benzodiazepine tranquilizers, acetylcholine synthesis, storage orrelease enhancers, acetylcholine postsynaptic receptor agonists,monoamine oxidase-A or -B inhibitors, N-methyl-D-aspartate glutamatereceptor antagonists, non-steroidal anti-inflammatory drugs,antioxidants, and serotonergic receptor antagonists.

In particular, the biologically active agent or compound may comprise atleast one compound selected from the group consisting of compoundsagainst oxidative stress, anti-apoptotic compounds, metal chelators,inhibitors of DNA repair such as pirenzepine and metabolites,3-amino-1-propanesulfonic acid (3APS), 1,3-propanedisulfonate (1,3PDS),secretase activators, [beta]- and 7-secretase inhibitors, tau proteins,neurotransmitter, /3-sheet breakers, antiinflammatory molecules,“atypical antipsychotics” such as, for example clozapine, ziprasidone,risperidone, aripiprazole or olanzapine or cholinesterase inhibitors(ChEIs) such as tacrine, rivastigmine, donepezil, and/or galantamine andother drugs and nutritive supplements such as, for example, vitamin B12, cysteine, a precursor of acetylcholine, lecithin, choline, Ginkgobiloba, acetyl-L-carnitine, idebenone, propentofylline, or a xanthinederivative, together with a binding peptide according to the inventionincluding antibodies, particularly monoclonal antibodies and activefragments thereof, and, optionally, a pharmaceutically acceptablecarrier and/or a diluent and/or an excipient and instructions for thetreatment of diseases.

In a further embodiment, the composition according to the invention maycomprise niacin or memantine together with a binding peptide accordingto the invention including antibodies, particularly monoclonalantibodies and active fragments thereof, and, optionally, apharmaceutically acceptable carrier and/or a diluent and/or anexcipient.

In still another embodiment of the invention compositions are providedthat comprise “atypical antipsychotics” such as, for example clozapine,ziprasidone, risperidone, aripiprazole or olanzapine for the treatmentof positive and negative psychotic symptoms including hallucinations,delusions, thought disorders (manifested by marked incoherence,derailment, tangentiality), and bizarre or disorganized behavior, aswell as anhedonia, flattened affect, apathy, and social withdrawal,together with the binding peptide according to the invention includingantibodies, particularly monoclonal antibodies and active fragmentsthereof, and, optionally, a pharmaceutically acceptable carrier and/or adiluent and/or an excipient.

Other compounds that can be suitably used in compositions in addition tothe binding peptide according to the invention, are those disclosed, forexample, in WO 2004/058258 (see especially pages 16 and 17) includingtherapeutic drug targets (page 36-39), alkanesulfonic acids andalkanolsulfuric acid (pages 39-51), cholinesterase inhibitors (pages51-56), NMDA receptor antagonists (pages 56-58), estrogens (pages58-59), non-steroidal anti-inflammatory drugs (pages 60-61),antioxidants (pages 61-62), peroxisome proliferators-activated receptors(PPAR) agonists (pages 63-67), cholesterol-lowering agents (pages68-75); amyloid inhibitors (pages 75-77), amyloid formation inhibitors(pages 77-78), metal chelators (pages 78-79), anti-psychotics andanti-depressants (pages 80-82), nutritional supplements (pages 83-89)and compounds increasing the availability of biologically activesubstances in the brain (see pages 89-93) and prodrugs (pages 93 and94), which document is incorporated herein by reference, but especiallythe compounds mentioned on the pages indicated above.

Proteinaceous pharmaceutically active matter may be present in amountsbetween 1 ng and 10 mg per dose. Generally, the regime of administrationshould be in the range of between 0.1 μg and 10 mg of the antibodyaccording to the invention, particularly in a range 1.0 μg to 1.0 mg,and more particularly in a range of between 1.0 μg and 100 μg, with allindividual numbers falling within these ranges also being part of theinvention. If the administration occurs through continuous infusion amore proper dosage may be in the range of between 0.01 μg and 10 mgunits per kilogram of body weight per hour with all individual numbersfalling within these ranges also being part of the invention.

Administration will generally be parenterally, e.g. intravenously.Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions and emulsions. Non-aqueous solventsinclude, without being limited to, propylene glycol, polyethyleneglycol, vegetable oil such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous solvents may be chosen from the groupconsisting of water, alcohol/aqueous solutions, emulsions or suspensionsincluding saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringers dextrose, dextrose and sodium chloride,lactated Ringers, or fixed oils. Intravenous vehicles include fluid andnutrient replenishers, electrolyte replenishers (such as those based onRingers dextrose) and others. Preservatives may also be present such as,for example, antimicrobials, antioxidants, chelating agents, inertgases, etc.

The pharmaceutical composition may further comprise proteinaceouscarriers such as, for example, serum albumin or immunoglobulin,particularly of human origin. Further biologically active agents may bepresent in the pharmaceutical composition of the invention dependent onits the intended use.

When the binding target is located in the brain, certain embodiments ofthe invention provide for the binding peptide according to the inventionincluding antibodies, particularly monoclonal antibodies and activefragments thereof, to traverse the blood-brain barrier. Certainneurodegenerative diseases are associated with an increase inpermeability of the blood-brain barrier, such that the binding peptideaccording to the invention including antibodies, particularly monoclonalantibodies or active fragment thereof can be readily introduced to thebrain. When the blood-brain barrier remains intact, several art-knownapproaches exist for transporting molecules across it, including, butnot limited to, physical methods, lipid-based methods, and receptor andchannel-based methods.

Physical methods of transporting the binding peptide according to theinvention including antibodies, particularly monoclonal antibodies, oractive fragment thereof across the blood-brain barrier include, but arenot limited to, circumventing the blood-brain barrier entirely, or bycreating openings in the blood-brain barrier. Circumvention methodsinclude, but are not limited to, direct injection into the brain (see,e.g., Papanastassiou et al., Gene Therapy 9: 398-406 (2002)) andimplanting a delivery device in the brain (see, e.g., Gill et al.,Nature Med. 9: 589-595 (2003); and Gliadel Wafers™, GuildfordPharmaceutical). Methods of creating openings in the barrier include,but are not limited to, ultrasound (see, e.g., U.S. Patent PublicationNo. 2002/0038086), osmotic pressure (e.g., by administration ofhypertonic mannitol (Neuwelt, E. A., Implication of the Blood-BrainBarrier and its Manipulation, Vols 1 & 2, Plenum Press, N. Y. (1989)),permeabilization by, e.g., bradykinin or permeabilizer A-7 (see, e.g.,U.S. Pat. Nos. 5,112,596, 5,268,164, 5,506,206, and 5,686,416), andtransfection of neurons that straddle the blood-brain barrier withvectors containing genes encoding the binding peptide or antigen-bindingfragment (see, e.g., U.S. Patent Publication No. 2003/0083299).

Lipid-based methods of transporting the binding peptide according to theinvention including antibodies, particularly monoclonal antibodies, oran active fragment thereof across the blood-brain barrier include, butare not limited to, encapsulating the binding peptide according to theinvention including antibodies, particularly monoclonal antibodies, oractive fragment thereof in liposomes that are coupled to activefragments thereof that bind to receptors on the vascular endothelium ofthe blood-brain barrier (see, e.g., U.S. Patent Application PublicationNo. 20020025313), and coating the binding peptide according to theinvention including antibodies, particularly monoclonal antibodies, oractive fragment thereof in low-density lipoprotein particles (see, e.g.,U.S. Patent Application Publication No. 20040204354) or apolipoprotein E(see, e.g., U.S. Patent Application Publication No. 20040131692).

Receptor and channel-based methods of transporting the binding peptideaccording to the invention including antibodies, particularly monoclonalantibodies, or active fragment thereof across the blood-brain barrierinclude, but are not limited to, using glucocorticoid blockers toincrease permeability of the blood-brain barrier (see, e.g., U.S. PatentApplication Publication Nos. 2002/0065259, 2003/0162695, and2005/0124533); activating potassium channels (see, e.g., U.S. PatentApplication Publication No. 2005/0089473), inhibiting ABC drugtransporters (see, e.g., U.S. Patent Application Publication No.2003/0073713); coating antibodies with a transferrin and modulatingactivity of the one or more transferrin receptors (see, e.g., U.S.Patent Application Publication No. 2003/0129186), and cationizing theantibodies (see, e.g., U.S. Pat. No. 5,004,697).

Single or repeated administrations of the binding peptide according tothe invention including antibodies, particularly monoclonal antibodies,or an active fragment thereof, or of a pharmaceutical compositionaccording to the invention may be provided to a subject over an extendedperiod of time. The duration of administration may be between 1 week andup to 12 month or more. During this time the binding peptide, antibodyor pharmaceutical composition may be administered once a week, onceevery two weeks, three weeks, four weeks, etc, or at a higher or lowerfrequency depending on the needs of the subject to be treated.

In a further embodiment the present invention provides methods and kitsfor the detection and diagnosis of tau-protein-associated diseases,disorders or conditions, including neurodegenerative diseases ordisorders such as tauopathies comprising a heterogenous group ofneurodegenerative diseases or disorders including diseases or disorderswhich show co-existence of tau and amyloid pathologies including, butnot limited to, Alzheimer's Disease, Creutzfeldt-Jacob disease, Dementiapugilistica, Down's Syndrome, Gerstmann-Sträussler-Scheinker disease,inclusion-body myositis, and prion protein cerebral amyloid angiopathy,traumatic brain injury and further of diseases or disorders which do notshow a distinct amyloid pathology including, but not limited to,amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam,Non-Guamanian motor neuron disease with neurofibrillary tangles,argyrophilic grain dementia, corticobasal degeneration, diffuseneurofibrillary tangles with calcification, frontotemporal dementia withparkinsonism linked to chromosome 17, Hallevorden-Spatz disease,multiple system atrophy, Niemann-Pick disease, type C,Pallido-ponto-nigral degeneration, Pick's disease, progressivesubcortical gliosis, progressive supranuclear palsy, Subacute sclerosingpanencephalitis Tangle only dementia, Postencephalitic Parkinsonism,Myotonic dystrophy. The pathological abnormalities may be caused by orassociated with the formation of neurofibrillary lesions, thepredominant brain pathology in tauopathy.

Further, the present invention provides methods and kits for diagnosinga predisposition to tau-protein-associated diseases, disorders orconditions, including neurodegenerative diseases or disorders such astauopathies comprising a heterogenous group of neurodegenerativediseases or disorders including diseases or disorders which showco-existence of tau and amyloid pathologies, or for monitoring minimalresidual disease in a patient or for predicting responsiveness of apatient to a treatment with a binding peptide according to the inventionincluding antibodies, particularly monoclonal antibodies and activefragments thereof, or a composition according to the invention and asdescribed herein. These methods include known immunological methodscommonly used for detecting or quantifying substances in biologicalsamples or in an in situ condition.

Diagnosis of a tau-protein-associated disease or condition or of apredisposition to an tau-protein-associated disease or condition in asubject in need thereof, particularly a mammal, more particularly ahuman, including neurodegenerative diseases or disorders such astauopathies comprising a heterogenous group of neurodegenerativediseases or disorders including diseases or disorders which showco-existence of tau and amyloid pathologies, may be achieved bydetecting the immunospecific binding of a binding peptide of theinvention, particularly of an antibody, particularly of a monoclonalantibody or an active fragment thereof, to an epitope of the tau proteinin a sample or in situ, which includes bringing the sample or a specificbody part or body area suspected to contain the tau protein into contactwith an antibody which binds an epitope of the tau protein, allowing theantibody to bind to the tau protein to form an immunologic complex,detecting the formation of the immunologic complex and correlating thepresence or absence of the immunologic complex with the presence orabsence of tau protein in the sample or specific body part or area,optionally comparing the amount of the immunologic complex to a normalcontrol value, wherein an increase in the amount of the immunologiccomplex compared to a normal control value indicates that the subject issuffering from or is at risk of developing an tau protein-associateddisease or condition.

Monitoring minimal residual disease in a subject, particularly a mammal,more particularly a human, following treatment with a binding peptideaccording to the invention including antibodies, particularly monoclonalantibodies and active fragments thereof, or a composition according tothe invention may be achieved by detecting the immunospecific binding ofa binding peptide of the invention, particularly of an antibody,particularly a monoclonal antibody or an active fragment thereof to anepitope of the tau protein in a sample or in situ, which includesbringing the sample or a specific body part or body area suspected tocontain the tau protein into contact with a binding peptide according tothe invention including antibodies, particularly monoclonal antibodiesand active fragments thereof, which binds an epitope of the tau protein,allowing the binding peptide according to the invention includingantibodies, particularly monoclonal antibodies and active fragmentsthereof, to bind to the tau protein to form an immunologic complex,detecting the formation of the immunologic complex and correlating thepresence or absence of the immunologic complex with the presence orabsence of tau protein in the sample or specific body part or area,optionally comparing the amount of said immunologic complex to a normalcontrol value, wherein an increase in the amount of said immunologiccomplex compared to a normal control value indicates that the subjectmay still suffer from a minimal residual disease.

Predicting responsiveness of a subject, particularly a mammal, moreparticularly a human, to a treatment with a binding peptide according tothe invention including antibodies, particularly monoclonal antibodiesand active fragments thereof, or a composition according to theinvention may be achieved by detecting the immunospecific binding of abinding peptide, particularly of a monoclonal antibody or an activefragment thereof to an epitope of the tau protein in a sample or insitu, which includes bringing the sample or a specific body part or bodyarea suspected to contain the tau protein into contact with a bindingpeptide according to the invention including antibodies, particularlymonoclonal antibodies and active fragments thereof, which binds anepitope of the tau protein, allowing the binding peptide according tothe invention including antibodies, particularly monoclonal antibodiesand active fragments thereof, to bind to the tau protein to form animmunologic complex, detecting the formation of the immunologic complexand correlating the presence or absence of the immunologic complex withthe presence or absence of tau protein in the sample or specific bodypart or area, optionally comparing the amount of said immunologiccomplex before and after onset of the treatment, wherein an decrease inthe amount of said immunologic complex indicates that said patient has ahigh potential of being responsive to the treatment.

Biological samples that may be used in the diagnosis of a tauprotein-associated disease or condition, for diagnosing a predispositionto a tau protein-associated disease or condition, includingneurodegenerative diseases or disorders such as tauopathies comprising aheterogenous group of neurodegenerative diseases or disorders includingdiseases or disorders which show co-existence of tau and amyloidpathologies, or for monitoring minimal residual disease in a patient orfor predicting responsiveness of a patient to a treatment with a bindingpeptide according to the invention including antibodies, particularlymonoclonal antibodies and active fragments thereof, or a compositionaccording to the invention and as described herein are, for example,fluids such as serum, plasma, saliva, gastric secretions, mucus,cerebrospinal fluid, lymphatic fluid and the like or tissue or cellsamples obtained from an organism such as neural, brain, cardiac orvascular tissue. For determining the presence or absence of the tauprotein in a sample, any immunoassay known to those of ordinary skill inthe art may be used such as, for example, assays which utilize indirectdetection methods using secondary reagents for detection, ELISAs andimmunoprecipitation and agglutination assays. A detailed description ofthese assays is, for example, given in Harlow and Lane, Antibodies: ALaboratory Manual (Cold Spring Harbor Laboratory, New York 1988 555-612,WO96/13590 to Maertens and Stuyver, Zrein et al. (1998) and WO96/29605.

For in situ diagnosis, the binding peptide according to the inventionincluding antibodies, particularly monoclonal antibodies and activefragments thereof, of the invention or any active and functional partthereof may be administered to the organism to be diagnosed by methodsknown in the art such as, for example, intravenous, intranasal,intraperitoneal, intracerebral, intraarterial injection such that aspecific binding between an antibody according to the invention with anepitopic region on the amyloid protein may occur. The bindingpeptide/antigen complex may conveniently be detected through a labelattached to the binding peptide according to the invention includingantibodies, particularly monoclonal antibodies, or a functional fragmentthereof or any other art-known method of detection.

The immunoassays used in diagnostic applications or in applications fordiagnosing a predisposition to a tau protein-associated disease orcondition, including neurodegenerative diseases or disorders such astauopathies comprising a heterogenous group of neurodegenerativediseases or disorders including diseases or disorders which showco-existence of tau and amyloid pathologies, or for monitoring minimalresidual disease in a patient or for predicting responsiveness of apatient to a treatment with a binding peptide according to the inventionincluding antibodies, particularly monoclonal antibodies and activefragments thereof, or a composition according to the invention and asdescribed herein typically rely on labelled antigens, binding peptides,or secondary reagents for detection. These proteins or reagents can belabelled with compounds generally known to those of ordinary skill inthe art including enzymes, radioisotopes, and fluorescent, luminescentand chromogenic substances including, but not limited to coloredparticles, such as colloidal gold and latex beads. Of these, radioactivelabelling can be used for almost all types of assays and with mostvariations. Enzyme-conjugated labels are particularly useful whenradioactivity must be avoided or when quick results are needed.Fluorochromes, although requiring expensive equipment for their use,provide a very sensitive method of detection. Binding peptides useful inthese assays are those disclosed claimed herein including antibodies,particularly monoclonal antibodies, polyclonal antibodies, and affinitypurified polyclonal antibodies.

Alternatively, the binding peptide according to the invention includingantibodies, particularly monoclonal antibodies and active fragmentsthereof, may be labelled indirectly by reaction with labelled substancesthat have an affinity for immunoglobulin, such as protein A or G orsecond antibodies. The binding peptide according to the inventionincluding antibodies, particularly monoclonal antibodies and activefragments thereof, may be conjugated with a second substance anddetected with a labelled third substance having an affinity for thesecond substance conjugated to the antibody. For example, the bindingpeptide according to the invention including antibodies, particularlymonoclonal antibodies and active fragments thereof, may be conjugated tobiotin and the binding peptide/biotin conjugate detected using labelledavidin or streptavidin. Similarly, the binding peptide may be conjugatedto a hapten and the binding peptide/hapten conjugate detected usinglabelled anti-hapten binding peptide.

Those of ordinary skill in the art will know of these and other suitablelabels which may be employed in accordance with the present invention.The binding of these labels to binding peptides or fragments thereof canbe accomplished using standard techniques commonly known to those ofordinary skill in the art. Typical techniques are described by Kennedy,J. H., et al., 1976 (Clin. Chim. Acta 70:1-31), and Schurs, A. H. W. M.,et al. 1977 (Clin. Chim Acta 57:1-40). Coupling techniques mentioned inthe latter are the glutaraldehyde method, the periodate method, thedimaleimide method, and others, all of which are incorporated byreference herein.

Current immunoassays utilize a double antibody method for detecting thepresence of an analyte, wherein, the antibody is labeled indirectly byreactivity with a second antibody that has been labeled with adetectable label. The second antibody is preferably one that binds toantibodies of the animal from which the monoclonal antibody is derived.In other words, if the monoclonal antibody is a mouse antibody, then thelabeled, second antibody is an anti-mouse antibody. For the antibody tobe used in the assay described herein, this label is preferably anantibody-coated bead, particularly a magnetic bead. For the antibody tobe employed in the immunoassay described herein, the label is preferablya detectable molecule such as a radioactive, fluorescent or anelectrochemiluminescent substance.

An alternative double antibody system, often referred to as fast formatsystems because they are adapted to rapid determinations of the presenceof an analyte, may also be employed within the scope of the presentinvention. The system requires high affinity between the antibody andthe analyte. According to one embodiment of the present invention, thepresence of the amyloid protein is determined using a pair ofantibodies, each specific for amyloid protein. One of said pairs ofantibodies is referred to herein as a “detector antibody” and the otherof said pair of antibodies is referred to herein as a “captureantibody”. The monoclonal antibody of the present invention can be usedas either a capture antibody or a detector antibody. The monoclonalantibody of the present invention can also be used as both capture anddetector antibody, together in a single assay. One embodiment of thepresent invention thus uses the double antibody sandwich method fordetecting amyloid protein in a sample of biological fluid. In thismethod, the analyte (amyloid protein) is sandwiched between the detectorantibody and the capture antibody, the capture antibody beingirreversibly immobilized onto a solid support. The detector antibodywould contain a detectable label, in order to identify the presence ofthe antibody-analyte sandwich and thus the presence of the analyte.

Exemplary solid phase substances include, but are not limited to,microtiter plates, test tubes of polystyrene, magnetic, plastic or glassbeads and slides which are well known in the field of radioimmunoassayand enzyme immunoassay. Methods for coupling antibodies to solid phasesare also well known to those of ordinary skill in the art. Morerecently, a number of porous material such as nylon, nitrocellulose,cellulose acetate, glass fibers and other porous polymers have beenemployed as solid supports.

The present invention also relates to a diagnostic kit for detecting tauprotein in a biological sample comprising a composition as definedabove. Moreover, the present invention relates to the latter diagnostickit which, in addition to a composition as defined above, also comprisesa detection reagent as defined above. The term “diagnostic kit” refersin general to any diagnostic kit known in the art. More specifically,the latter term refers to a diagnostic kit as described in Zrein et al.(1998).

It is still another object of the present invention to provide novelimmunoprobes and test kits for detection and diagnosis of tauprotein-associated diseases and conditions, comprising binding peptidesaccording to the present invention. For immunoprobes, the bindingpeptides are directly or indirectly attached to a suitable reportermolecule, e.g., an enzyme or a radionuclide. The test kit includes acontainer holding one or more binding peptides according to the presentinvention and instructions for using the binding peptides for thepurpose of binding to tau antigen to form an immunologic complex anddetecting the formation of the immunologic complex such that presence orabsence of the immunologic complex correlates with presence or absenceof tau protein.

EXAMPLES Example 1 Generation and Screening of Hybridomas and Antibodies

The objective of this study was to generate and screen anti-Tau mAbs(monoclonal antibodies). Hybridomas were generated by fusion of tauvaccine immunized mouse spleen with a myeloma cell line. The hybridomaswere assessed for reactivity against both phosphorylated andnon-phosphorylated full-length Tau protein, as well as thephosphorylated and non-phosphorylated Tau antigenic peptides used in thevaccine preparation. Hybridoma screening was also performed forreactivity of hybridomas supernatant for tau tangles usingimmunochemistry on Tau transgenic mouse brain slices.

1.1 Methods 1.1.1 Fusion

A wild type C57BL/6 mouse vaccinated with ACI-33 (Tau5-20 [pY18]) wasused for hybridoma production. The mouse was boosted with ACI-33 vaccineon day 0 then again on day 4 and the fusion was performed on day 7.173×10⁶ (ACI-33), splenocytes from the immunized mouse were fused withSP2-O-Ag14 myeloma cells at a ratio of 5 splenocytes/1 myeloma cell.

A wild type C57BL/6 mouse vaccinated with ACI-35 (Tau393-408 [pS396,pS404]) was used for hybridoma production. The mouse was boosted withACI-35 vaccine on day 0 then again on day 4 and the fusion was performedon day 6×10⁷ (ACI-35), splenocytes from the immunized mouse were fusedwith 2×10⁷ SP2-O-Ag14 myeloma cells at a ratio of 3 splenocytes/1myeloma cell.

A wild type C57BL/6 mouse vaccinated with ACI-36 (Tau401-418[pS404/S409]) was used for hybridoma production. The mouse was boostedwith ACI-36 vaccine on day 0 then again on day 4 and the fusion wasperformed on day 7. 84×10⁶ splenocytes from the immunized mouse werefused with SP2-O-Ag14 myeloma cells at a ratio of 5 splenocytes/1myeloma cell.

A wild type C57BL/6 mouse vaccinated with ACI-41 (mix of Tau206-221[pT212/pS214] and Tau196-211 [pS202/pT205]) was used for hybridomaproduction. The mouse was boosted with ACI-41 vaccine on day 0 thenagain on day 4 and the fusion was performed on day 8. 162×106splenocytes from the immunized mouse were fused with SP2-O-Ag14 myelomacells at a ratio of 5 splenocytes/1 myeloma cell.

The four fusions resulted in 8×96 well plates and the clones were nameaccording to the plate (1-8) then the row (A-G) and finally the column(1-12).

1.1.2 Screening Method to Select Clones

The 8×96 well plates were first screened twice for IgG expression.Positive expressing clones were then transferred in 24 well plates andcell supernatants (=clones) of growing cells were tested in a Tau ELISAscreen and a immunohistochemistry TAUPIR screen. Positive supernatantsin ELISA and/or TAUPIR were transferred to T25 flasks and clones werescreened again for IgG expression in aTau ELISA screen and TAUPIRscreen.

1.1.3 IgG Screen

Elisa plates were coated with 50 ul/well of anti-mouse IgG antibody (CERGroupe, Marloie, Belgium) in coating buffer for 16 hrs at 4° C. Afterwashing plates with PBS/Tween 100 ul/well of a blocking solution wasapplied for 1 hr at RT. 50 ul of undiluted hybridoma supernatant wereincubated for 1 hr at RT. After a washing step, a mix of the HorseRadishPeroxydase (HRP)-conjugated anti-mouse IgG1, IgG2a, IgG2b and IgG3 (AbSerotec, Raleigh, N.C., USA) was applied on the plates for 1 hr at RT.After a final washing, detection was performed with TMB(3-3′,5,5′-tetramethylbenzidine), the phosphatase substrate for HRP, andplates were read at 405 nm using an ELISA plate reader. Results areexpressed as O.D. (Optical Density).

1.1.4 Hybridomas Tau ELISA Screen

Hybridomas ELISA screen was performed on pTau peptide (ACI-33, T1.5:Tau5-20 [pY18]; ACI-35, T3.5: Tau393-408[pS396/pS404]; ACI-36, T4.5:Tau401-418 [pS404/S409]; ACI-41, T8.5: Tau206-221 [pT212/pS214] andT9.5: Tau196-211 [pS202/pT205] PolyPeptide Laboratories, HiHerød,Denmark), corresponding Tau peptide (ACI-33, T1.6: Tau5-20; ACI-36,T4.6: Tau401-4; ACI-41, T8.6: Tau206-221 and T9.6: Tau196-211,PolyPeptide Laboratories, HiHerød, Denmark), phosphorylated full-length(441aa) Tau protein (pTau protein, Vandebroek et al., 2005) andfull-length (441aa) Tau protein (Tau protein, SignalChem, Richmond,Canada). Finally Bovine Serum Albumin (BSA) was used as negativecontrol.

Plates were coated with 10 μg/ml of corresponding Tau peptide and 1μg/ml of corresponding Tau protein overnight at 4° C. After washing eachwell with PBS-0.05% Tween 20 and blocking with 1% BSA in PBS-0.05% Tween20, undiluted hybridoma supernatant or medium negative control wereadded to the plates and incubated at 37° C. for 2 hours. After washingplates were incubated with an alkaline phosphatase (AP)-conjugatedanti-mouse IgG total antibody (Jackson Laboratories, Baltimore, Pa.,USA) for 2 hours at 37° C. After washing plates were incubated with pNPP(para-nitro-phenyl-phosphate), the phosphatase substrate for AP, andread at 405 nm using an ELISA plate reader. Results are expressed asO.D. (Optical Density).

1.1.5 Hybridomas IHC Screen: Binding of Anti-Tau Antibodies to Tanglesin Brain Sections from Transgenic Mice (TAUPIR)

TAUPIR experiments were done according to protocol from EXAMPLE 3.1.2.

1.1.6 T25 Flasks IgG Screen

Elisa plates were coated with 5 ug/ml of anti-mouse IgG F(ab′)2 fragmentspecific antibody (Jackson Laboratories, Baltimore, Pa., USA) incarbonate-bicarbonate coating buffer pH 9.6 (Sigma, Buchs, Switzerland)overnight at 4° C. After washing plates, undiluted hybridomasupernatant, positive control IgG1 antibody (6E10 at 1 ug/ml: Covance,Emeryville, Calif., USA) or negative control (culture medium alone) wereincubated for 1 hr at RT. After a washing step, the secondaryAP-conjugated goat anti-mouse IgG (subclasses 1+2a+2b+3) Fcγ fragmentspecific antibody (Jackson Laboratories, Baltimore, Pa., USA) wasincubated on the plates for 2 hrs at 37° C. After a final washing,detection was performed with pNPP (para-nitro-phenyl-phosphate), thephosphatase substrate for AP, and plates were read at 405 nm using anELISA plate reader. Results are expressed as O.D. (Optical Density).

1.2 Results 1.2.1 ACI-33 Hybridomas

The cell supernatants from the 8×96 well plates resulting from thefusion were screened for production of IgG. In the 768 wells (8×96wells) tested 277 wells were positive for IgG expression and weretransferred to 24 wells plates. In the 24 well plates 79 clones weregrowing and supernatant from those cells were analysed. Positive cloneswere further transferred in T25 flasks and supernatants screened for IgGproduction, ELISA and TAUPIR (Table 2).

The clone 6C10 was the only one positive in the 3 screens and wasselected for subcloning.

1.2.2 ACI-36 Hybridomas

The cell supernatants from the 8×96 well plates resulting from thefusion were screened for production of IgG. In the 768 wells (8×96wells) tested 333 wells were positive for IgG expression and weretransferred to 24 wells plates. In the 24 well plates 75 clones weregrowing and supernatant from those cells were analysed. Positive cloneswere further transferred in T25 flasks and supernatants screened for IgGproduction, ELISA and TAUPIR (Table 3).

In order to select clones for the next steps a ranking of allsupernatants positives for IgG/ELISA/TAUPIR screens was performed basedon the ELISA and TAUPIR results. Ranking the ELISA and TAUPIR resultswas performed as explained in the methods section. TAUPIR staining wasalmost identical for the five first clones and this corresponded to theELISA results. 4C12 was discarded as it was found in the same plate as4C1 which increased the likelihood of the 2 clones being the same(recognizing the same epitope). The best 4 clones selected were 3A8,2B6, 4C1 and 6H1. The other 6 clones (4C12, 2G1, 2F9, 7D6, 3B9, 4E12)were kept as back-up.

A ranking of the 10 clones that showed positivity in ELISA screen andTAUPIR screen was performed to select the best ones (Table 4).Highlighted in grey are the best 5 clones.

1.2.3 ACI-41 Hybridomas

The cell supernatants from the 8×96 well plates resulting from thefusion were screened for production of IgG. In the 768 wells (8×96wells) tested 215 wells were positive for IgG expression and weretransferred to 24 wells plates. In the 24 well plates 81 clones weregrowing and supernatant from those cells were analysed. Positive cloneswere further transferred in T25 flasks and supernatants screened for IgGproduction, ELISA and TAUPIR (table 5).

The clones 5D10 and 7C2 were the only ones positive in the 3 screens andwere selected for subcloning. The clone 5D10 binds only the peptideT8.5, while the clone 7C2 binds to the two peptides of the ACI-41vaccine (T8.5 and T9.5) (see FIG. 10 in PCT applicationPCT/EP2010/054418).

The subclone 5D10A4 originating from 5D10 was specific for pTau peptide.

1.3. Conclusion

The antibodies generated have shown high specificity to pTau peptideswith only marginal binding to non-phosphorylated peptides.

From the 4 fusions (ACI-33, ACI-36, ACI-35 and ACI-41), a total of 16clones were deposited at DSMZ (table 1) and selected for furthersubcloning.

The positive motherclones mentioned above were further cultivated in 96well plates, then 24 well plates and finally T25 flasks. At each stage,the supernatants of the hybridoma clones were screened by ELISA, Taupirand Western Blot.

Example 2 Cloning of Antibody Light Chain and Heavy Chain VariableRegions

Antibody heavy and light variable region genes from the hybridoma cellsare cloned and the DNA sequences and location of the complementaritydetermining regions (CDRs) determined as well as the antibodies bindingfeatures.

Total RNA was prepared from 3×10⁶ hybridoma cells (1 vial) using theQiagen RNeasy mini kit (Cat No: 74104). RNA was eluted in 50 mL waterand checked on a 1.2% agarose gel.

V_(H) and V_(K) cDNAs were prepared using reverse transcriptase with IgGand kappa constant region primers. The first strand cDNAs were amplifiedby PCR using a large set of signal sequence primers. The amplified DNAswere gel-purified and cloned into the vector pGem® T Easy (Promega). TheV_(H) and V_(K) clones obtained were screened for inserts of theexpected size. The DNA sequence of selected clones was determined inboth directions by automated DNA sequencing. The locations of thecomplementarity determining regions (CDRs) in the sequences weredetermined with reference to other antibody sequences (Kabat E A et al.,1991).

Example 3 Binding Studies I

The objective was to measure the phospho-Tau (pTau) binding of theantibodies generated from subcloned hybridomas derived from miceimmunized with the tau liposomal vaccines. To test this, anenzyme-linked immunosorbant assay (ELISA) was used to measure thebinding of the purified antibodies to both phosphorylated andnon-phosphorylated full-length Tau protein, as well as thephosphorylated and non-phosphorylated Tau antigenic peptides used forthe liposomal vaccine preparation.

The screening was completed by two other methods. Immunohistochemistry(IHC) on brain sections from a Tau transgenic animal (TAUPIR) using ananti-tau antibody as the primary antibody was done. Additionally, awestern blot (WB) on brain protein homogenates from Tau transgenic micewas performed, using an anti-tau antibody as the blotting antibody.

3.1 Methods 3.1.1 Phospho-Tau Binding Assay

The anti-phospho Tau antibodies (mouse IgG3 isotype) were generated fromliposomal tau vaccinated mice. The liposomal vaccines are phosphorylatedpreparations of a phospho-Tau (pTau) peptide. The hybridoma sub-clonesproducing the anti-tau antibodies were selected by limiting dilutionfrom the mother-clones. Isotyping was done to indicate the presence of asingle isotype clone. The antibodies was produced in roller-bottles,purified by affinity chromatography, subjected to sterile 0.22 μmfiltration, and quantified. To test the binding of the antibody to Tauand pTau, an ELISA assay was used. Briefly, Nunc MaxiSorp 96-well plates(Nunc, Roskilde, Denmark) were coated with 1 μg/mL of full-length (441aa) Tau protein (SignalChem, Richmond, Canada) or phosphorylatedfull-length (441 aa) Tau protein (Vandebroek et al., 2005).Additionally, plates were coated with 10 μg/mL of the Tau-derivedpeptide. To test for cross-reactivity to Tau and pTau sequences thatwere not used in the vaccine preparation, plates were coated with 10μg/mL of the following peptides: Tau5-20 (phosphorylated or not on Y18),Tau393-408 (phosphorylated or not on S396 and S404), Tau401-418(phosphorylated or not on S404 and S409), Tau206-221 (phosphorylated ornot on T212 and S214), and Tau196-211 (phosphorylated or not on S202 andT205). Coating was done overnight in phosphate-buffered saline (PBS) at4° C. Plates were washed thoroughly with 0.05% Tween20/PBS and thenblocked with 1% bovine serum albumin (BSA) in 0.05% Tween20/PBS for 1 hrat 37° C. The antibody being tested was then added in an 8 or 16two-fold dilution series between 20 and 0 μg/mL, and allowed to incubatefor 2 hr at 37° C. Plates were then washed as described previously, andAP-conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearchLaboratories, Suffolk, England) was added at 1/6000 dilution in 0.05%Tween20/PBS for 2 hr at 37° C. After washing, plates were incubated withp-nitrophenyl phosphate disodium hexahydrate (pNPP; Sigma-Aldrich,Buchs, Switzerland) phosphatase substrate solution, and read at 405 nmfollowing 2 or 16 hr incubation using an ELISA plate reader. Results areexpressed as optical density (O.D.).

3.1.2 Binding of Anti-Tau Antibody to Tau Tangles in Brain Sections froma Tau Transgenic Animal (TAUPIR)

Brain slices used were from old (>18 months old) double transgenic biGT(GSK-3β transgenic mice crossed with TPLH mice, containing the longestisoform (441aa) of human Tau with the P301L mutation) transgenic mice.Additionally, sections from Tau knock-out mice (TKO; 6 months old) werealso used. Brain sections were washed for 5 min in PBS then incubatedfor 15 min at RT in 1.5% H₂O₂ in PBS:MeOH (1:1) to block endogenousperoxidase activity. After washing the sections 3 times in PBST(PBS/0.1% TritonX100) they were incubated for 30 min at RT in PBST+10%FCS (fetal calf serum) blocking solution. The incubation with theanti-Tau antibody being tested was done overnight at 4° C. at indicateddilutions in PBST/10% FCS. Sections were next washed 3 times in PBSTbefore incubation with an HRP-conjugated goat anti-mouse (purchased fromDako, Glostrup, Denmark) secondary antibody in PBST/10% FCS for 1 hourat RT. Prior to detection, sections were washed 3 times with PBST andincubated in 50 mM Tris/HCl pH7.6 for 5 min. Detection was done byincubating the sections for 3 min in Diaminobenzidine (DAB: 1 tablet in10 ml of 50 mM Tris.HCl+3 ul H₂O₂ 30%; MP Biomedicals, Solon, Ohio,USA). The reaction was stopped by washing the sections 3 times in PBST.Sections were then transferred onto silanized glass-plates and air-driedon warm-plate at 50° C. for 2 hours. Counterstaining was done usingincubation with Mayers hematoxylin (Fluka Chemie, Buchs, Switzerland)for 1 min, followed by a washing step for 4 min in running tap-water.Sections were dehydrated by passing in 50%, 70%, 90% and twice in 100%ethanol bath then in Xylol 2 times for 1 min. Finally sections weremounted with DePeX (BDH Chemicals Ltd., Poole, England) under glasscover-slips.

Additionally, hybridoma supernatants at 1/10 dilution (allACI-35-derived antibodies shown in Table 1) were used to blot membranescontaining SDS-PAGE separated brain homogenate proteins from Tautransgenic mice, wild-type mice, or Tau knock-out mice.

3.1.3. Binding of Anti-Tau Antibody to Tau Tangles in Brain Sectionsfrom AD and Tauopathy Patients (TAUPIR)

The assay for the immunoreaction of the anti-pTau antibodyACI-36-3A8-Ab1 to pTau in human brain was done by TAUPIR. Brain paraffinsections were de-paraffinized by passing in Xylol 2 times for 5 min and2 times for 1 min in 100% EtOH, followed by 1 min wash in 90%, 70%, and50% EtOH and distilled water, followed by 2 times 5 min washes in PBS.For antigen retrieval, sections were treated by heating for 10 min in0.01 M citric acid solution in water (pH 6.0) and cooled down for 20min. Sections were incubated for 15 min at RT in 1.5% H₂O₂ in PBS:MeOH(1:1) to block endogenous peroxidase activity. After washing thesections 3 times in PBST (PBS/0.05% Tween-20), they were incubated for30 min at RT in PBST+10% fetal calf serum (FCS) as blocking solution.The incubation with the primary anti-pTau antibody ACI-36-3A8-Ab1 (410ng/mL in blocking buffer) was done overnight at 4° C. Sections were thenwashed 3 times in PBST before incubation with HRP-conjugated goatanti-mouse secondary antibody (Dako, Glostrup, Denmark) diluted 1/500 inPBST/10% FCS, for 1 hour at RT. Prior to detection, sections were washed3 times with PBS and incubated in 50 mM Tris/HCl pH 7.6 for 5 min.Detection was done by incubating the sections for 3 min indiaminobenzidine (DAB: 1 tablet in 10 mL of 50 mM Tris-HCl+3 μL H₂O₂30%; MP Biomedicals, Solon, Ohio, USA). The reaction was stopped bywashing the sections 3 times in PBS. Counterstaining was done byincubating with Mayer's hematoxylin (Fluka Chemie, Buchs, Switzerland)for 1 min, followed by washing for 4 min in running tap-water. Sectionswere dehydrated by passing through 50%, 70%, 90% and twice in 100%ethanol baths, followed by Xylol for 2 times 1 min.

Finally, sections were mounted with DePeX (BDH Chemicals Ltd., Poole,England) under glass cover-slips. Stained sections were examined bywhite light microscopy and digital images taken with a 3CCD camera(Leica, Wetzlar, Germany). Images were captured and analyzed usingdedicated software (IM500, Leica). Images are shown at 20×1.6magnification.

3.1.4. Western Blot (WB)

Binding of the test antibody to pTau in the brain extract fromtransgenic animal was done by WB. Brain homogenization from wild-typeFVB, TPLH, biGT and TKO mice was done in the following buffer: 25 mMTris/HCl pH7.6, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 30 mM NaF, 0.2 mMNa₃VO₄, 1 nM Okadaic acid, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5mM Na4P2O7, 1 tablet complete protease inhibitor cocktail (CPIC) per 12ml total. To obtain total brain homogenate the brain was homogenized onice in 1 vol/weight hemisphere (ml/g) with a motor-driven potter-likeglass tube/teflon pestle at 700 rpm. Total brain homogenates werediluted by half in sample buffer (125 mM Tris/HCl pH6.8, 4% (w/v) sodiumdodecyl sulfate (SDS), 20% glycerol, 0.01% bromophenol blue and 5%beta-mercapto-ethanol), then heated rapidly to 95° C. Samples were kept5 min, diluted 1/4 in sample buffer, heated again to 95° C. and thencooled down and spun at 14.000 rpm for 5 min to clear debris that werenot solubilized. Supernatants were collected and loaded onto a SDS-PAGEgel. The transfer to nitrocellulose membrane (Hybond-ECL) was done intransfer buffer (25 mM Tris pH 8.6, 190 mM Glycine, 20% methanol).Membranes were transferred to the blocking solution (0.1% Tween in TBS(50 mM Tris.HCl, pH7.6, 150 mM NaCl, and 5% dry-milk powder) prior toovernight incubation at 4° C. with the test antibody diluted in theblocking solution. Incubation with secondary antibody HRP-conjugatedgoat anti-mouse (Dako, Glostrup, Denmark) diluted 1/10,000 in blockingsolution was performed at RT for 1 hour. Detection was done using theECI Western Blotting Detection Reagents from GE Healthcare.

3.2 Results

3.2.1 ELISA Assays and TAUPIR Using Brain Sections from Tangle PositiveTau Transgenic Mice

The binding of antibodies were measured against the phosphorylated Taupeptide used as the immunogen, and against the phosphorylatedfull-length human Tau protein. This is the longest isoform of human Tauprotein consisting of 441 amino acids. The correspondingnon-phosphorylated peptide and full-length human Tau protein were alsoincluded. As indicated in the table 6 antibodies demonstrated highbinding to the phosphorylated Tau peptide, with only limited or nobinding to the phosphorylated full-length human Tau protein. No bindingwas observed to the corresponding non-phosphorylated Tau peptide or tothe non-phosphorylated full-length human Tau protein. This demonstrateshigh binding of anti-tau antibodies to phosphorylated human Taupeptides.

To test for non-specific binding to other phosphorylated andnon-phosphorylated Tau sequences, the antibody was tested for binding tofive phospho and non-phospho Tau peptides one of which was used as theantigen peptide sequence. No cross-reactivity to phospho or non-phosphoTau peptides, other than the peptide used in the vaccine was observed,even at high concentrations of peptide.

The binding of anti-tau antibodies to pTau in brains of Tau transgenicmice was evaluated by TAUPIR staining (FIG. 1) and by WB (FIG. 1).Antibodies demonstrated binding to Tau tangles and neuropil threadspresent in cortex and hippocampus in brains of Tau transgenic (biGT)mice. The antibody dilutions used for the TAUPIR ranged from 0.05 to0.0033 ug/mL. Anti-tau antibodies were also used as a primary antibodyin a WB using total brain homogenates from wild-type FVB, TPLH, biGT andTKO mice, and separated by SDS-PAGE. Two commercial anti-pTau antibodieswere used as controls, MC1 and Tau5. All anti-tau antibodies bound topTau present in brains of Tau transgenic mice. Blotting

On membranes containing SDS-PAGE separated protein homogenates from Tautransgenic mice, wild-type mice, and Tau knock-out mice, all ACI-35antibodies (disclosed in Table 1) bound to protein bands havingidentical 46 kDa migration pattern as Tau and pTau (data not shown).

3.2.2 TAUPIR Study in Brain Sections from AD and Tauopathy Patients

The ability of antibody ACI-36-3A8-Ab1 to bind to Tau-aggregates, lodgedin human brain sections from subjects with diagnosed tauopathies,including AD, FAD, AGD, FTDP-17, CBD, and PSP, was examined by TAUPIRimmunohistochemistry (FIG. 2). The anti-pTau antibody ACI-36-3A8-Ab1bound to pTau containing neurofibrillary tangles (NFTs), neuropilthreads in human brain sections, and other forms of pTau accumulationspresent in neurons and in glial cell-types. More specifically,ACI-36-3A8-Ab1 prominently stained NFTs, neuropil threads, anddystrophic neurites surrounding amyloid plaques in AD brains, which wasreadily apparent in the subjects diagnosed with AD and FAD. In brainsections from AGD, ACI-36-3A8-Ab1 stained both NFTs and neuropilthreads, with multiple argyrophilic grains/granules clearly visible(FIG. 2,). Staining of brain sections from PSP with ACI-36-3A8-Ab1showed NFTs, neuropil threads, and dystrophic neurites. Additionally,Pick body-like inclusions and tufted pTau positive astrocytes wereclearly noted, being an abundant feature in PSP, where pTau stainingextends throughout the cell, including in distal processes. In FTDP-17,the staining pattern also illustrated the known heterogeneity of thedisease, with not only NFTs but also achromatic “ballooned” neuronsdetected. The ACI-36-3A8-Ab1 antibody also stained swollen achromaticneurons that were faintly Tau-positive, the main characteristic of CBD.Another prominent pathological feature of CBD, i.e. oligodendroglialinclusions, called coiled bodies, were also well detected by theACI-36-3A8-Ab1 antibody. No staining was detected in an AT8-negativecontrol subject whereas weak staining was identified in an AT8-positivecontrol subject.

Using TAUPIR on human brain sections from subjects previously diagnosedwith different forms of tauopathy, the anti-pTau antibody ACI-36-3A8-Ab1demonstrated good binding to various known pTau-rich pathologicalfeatures present in the brains of these subjects.

Example 4 Binding Studies II

The objective of the study was to determine the binding affinity betweenanti-tau antibodies and the phospho-tau peptide using Surface PlasmonResonance (SPR). Phospho-tau peptide correspond to the peptide sequenceused in the vaccine preparation to generate the anti-tau antibody. Tostudy this interaction, phosphopeptides were immobilized to the surfaceof a sensor chip and the binding monitored in real-time using SPR uponpassing antibody over the chip.

4.1 Methods 4.1.1 SPR Binding Assay

All SPR experiments were carried out on a Biacore X instrument (GEHealthcare). Reagents for immobilization (EDC, NHS and Ethanolamine),sensor chip CM5 (carboxymethyl dextran) as well as running buffer HBS-EPwere purchased from GE Healthcare. Phospho-tau peptide were solubilizedin PBS/sodium acetate buffer (10 mM, pH 5.0) in a 1:1 (v/v) ratio togive a final peptide concentration of 250 μg/ml. This peptide solutionwas then coupled via to flow cell (fc) 2 of a CM5 sensor chip that waspreactivated using EDC/NHS. After coupling, Ethanolamine was passed overthe surface and giving a final immobilization level of 218 RUs. Fiveconcentrations of the anti-tau antibodies were assayed by serialdilutions using running buffer. Injections were performed starting fromthe lowest concentration and were passed over both fc 1 and 2 at a flowrate of 30 μL/min for 180 s. Flow cell 1 was underivatized and responseswere subtracted from fc 2 to correct for instrument noise and bulkrefractive changes. After injection was finished, the surfaces werewashed immediately with running buffer for 300 s. To remove remainingbound antibody from the chip, surface regeneration was performed byinjecting a pulse (typically 3 μl) of 8 mM NaOH in water containing 1MNaCl. Kinetic analysis was performed using algorithms for numericalintegration and global analysis using BIAevaluation 3.0. The sensogramsobtained for injections of antibody at different concentrations wereoverlaid and the baselines adjusted to zero. For curve fitting, all datawere fit simultaneously to a 1:1 homogeneous (Langmuir) model.

Alternatively, immobilized biotinylated T3 peptide (T3.30) wasimmobilized to a Streptavidin Biacore SA chip (GE Healthcare) using aBiacore X instrument. Antibodies were diluted in HBS-EP running buffer(GE Healthcare) and injected at 50 ul/min for 120 s followed by 100 sdissociation. Surface regeneration was performed using a pulse (1-3 ul)of 16 mM NaOH. Fitting was performed using BIAevaluation and assuming a1:1 Langmuir binding interaction.

Peptides Used

T1.5 H-K(Ac)K(Ac)-RQEFEVMEDHAGTY[PO3H2]GL- lot AW11309D K(Ac)K(Ac)-NH2T4.5 H-K(Ac)K(Ac)-GDTS[PO3H2]PRHLS[PO3H2]NVSSTGSID- lot CF09168K(Ac)K(Ac)-NH2 T3.30 Biotin-LC linker- lot MI89P9-P12-GVYKS[PO3H2]PVVSGDTS[PO3H2]PRHL-NH2 2

4.2 Results

The binding of the anti-tau antibodies to the phosphorylated Tau peptidewas monitored in real-time using SPR. Analyses of the association anddissociation phases of antibody binding could be used to determine theassociation rate constant (k_(a)), dissociation rate constant (k_(d)) aswell as dissociation constant K_(D). Antibody ACI-33-6C10-Ab1 bindsspecifically to peptide T1.5 over the non-derivatized carboxymethyldextran surface in the range 3.7→367 nM of antibody. Kinetic analyses ofthe sensograms revealed a fast association rate constant of 9.46×10⁵M⁻¹s⁻¹ and a dissociation rate constant of 3.27×10⁻³ s⁻¹ (Table 7). Thedissociation constant K_(D) was determined therefore to be 3.46 nMshowing that the antibody recognizes the phosphopeptide T1.5 with veryhigh affinity. All tested antibodies displayed a high affinity to theirrespective phosphopeptides used for immunization and hybridomageneration, but they displayed little affinity to non-phosphopeptides.

Example 5 Epitope Mapping of Anti pTau Antibodies 5.1 Methods

Epitope mapping of anti-phospho Tau mouse monoclonal antibodies wasperformed by ELISA using different phospho and non-phospho peptidelibraries. The amino acid sequences of peptide libraries used are shownin Table 8. Each library consisted of short biotinylated peptidesspanning phospho and non-phospho sequences present in the peptidevaccine. Peptide libraries were purchased from ANAWA Trading SA. Epitopemapping was done according to the manufacturer's (Mimotopes)instructions. Briefly, streptavidin coated plates (NUNC) were blockedwith 0.1% BSA in phosphate-buffered saline (PBS) overnight at 4° C.After washing with PBS-0.05% Tween 20, plates were coated for 1 hr at RTwith the different peptides from each library, diluted in 0.1% BSA, 0.1%sodium azide in PBS to a final concentration of 10 μM. After washing,plates were incubated for 1 hr at RT with the antibody to be testeddiluted to 40 ng/ml in 2% BSA, and 0.1% sodium azide in PBS. Plates werewashed again and incubated with AP-conjugated anti-mouse IgG secondaryantibody (Jackson ImmunoResearch Laboratories, Suffolk, England) at1/6000 dilution for 1 hr at RT. After a final wash, plates wereincubated with p-nitrophenyl phosphate disodium hexahydrate (pNPP;Sigma-Aldrich, Buchs, Switzerland) phosphatase substrate solution, andread at 405 nm following 2 hr incubation using an ELISA plate reader.Binding was considered positive if the optical density (O.D.) was atleast 2-times over background O.D.

5.2 Results

As a result of the epitope mapping experiments, epitopes could beidentified including the required phosphorylated amino acid residue (seetable 9) to which the antibodies disclosed herein specifically bind.

-   -   Tau aa 15-20, with requirement for pY18 (6C10F9C12A11;        6C10E5E9C12)    -   Tau aa 405-412, with requirement for pS409 (6H1A11C11; 6H1G6E6)    -   Tau aa 405-411, with requirement for pS409 (2B6A10C11; 2B6G7A12;        3A8A12G7; 3A8E12H8)    -   Tau aa 208-218, with requirement for pT212 and pS214        (7C2(1)F10C10D3)    -   Tau aa 393-401, with requirement for pS396 (A4-2A1-18;        A4-2A1-40)    -   Tau aa 396-401, with requirement for pS396 (A4-4A6-18)    -   Tau aa 394-400, with requirement for pS396 (A6-1D2-12)    -   Tau aa 402-406, with requirement for pS404 (A6-2G5-08)    -   Tau aa 393-400, with requirement for p396 (A6-2G5-30; A6-2G5-41)

Example 6 1-Week Passive Immunization of Tau Transgenic Mice 6.1.Methods

For all in vivo studies, Tau transgenic mice were used and administeredthe treatment antibodies as shown in the Table below.

Transgenic Mice and Antibodies Used for In Vivo Studies

Age of Number mice at Study of Study Tau transgenic study start durationAntibodies Doses i.p. no. model (months) (weeks) administered (mg/kg)administrations Readout 1 TMHT 6.3 1 ACI-36-2B6- 0*, 3 or 10 2 MSD,(hTau^(V337M/R406W)) Ab1 IHC, WB ACI-36-3A8-    0 or 3 Ab1 2 TMHT 4.2 4ACI-36-2B6- 0, 1 or 3 4 MSD, (hTau^(V337M/R406W)) Ab1 IHC, WB, or MWMACI-36-3A8- Ab1 3 TMHT 3.0 12 ACI-36-2B6- 0, 1 or 3 13 MSD,(hTau^(V337M/R406W)) Ab1 IHC, WB, or MWM ACI-36-3A8- Ab1 4 biGT 4.5 12ACI-36-2B6- 0, 1 or 3 13 WB (hTau^(P301L) × Ab1 hGSK3β) or ACI-36-3A8-Ab1 *vehicle control for all of the studies; intraperitoneally (i.p.)

6.1.1. Mice and Treatments

Female and male 6.3 months old (±3 days) Tg mice over-expressing thefull-length human TAU isoform TAU441, bearing the missense mutationsV337M and R406W under the control of murine Thy-1 promoter (TMHT mice),were used for Study no. 1 (see Table above). Mice were euthanized 1 dayfollowing the last administration to determine TAU pathology in thebrain.

6.1.2 Animal Identification and Housing

In the course of tail tipping for genotyping, animals were numberedconsecutively by classical earmarking. All animals were re-genotypedprior to the start of the study. Mice were kept according to the JSWStandard Operating Procedures based on international standards. Animalswere housed in individual ventilated cages on standardized rodentbedding supplied by Rettenmaier®. The temperature was maintained atapproximately 24° C. and the relative humidity was maintained between 40to 70%. Animals were housed under a constant light-cycle (12 hourslight/dark). Dried, pelleted standard rodent chow (Altromin®) and normaltap water were available to the animals ad libitum. Each individualanimal was checked regularly for any clinical signs that were noted inthe individual animal datasheet.

6.1.3 In Vivo Bleedings

Seven days before the first immunization, in vivo bleedings wereperformed by mandibular sampling from the facial vein/artery. The bloodsamples are a mixture of venous and arterial blood. To get plasma, bloodwas collected in heparin tubes and centrifuged (1000×g, 10 minutes, roomtemperature). Plasma was frozen in two aliquots until used.

6.1.4. Immunohistochemical (IHC) Quantitation

All cryo-frozen brain hemispheres were analyzed. 15 cryo-sections perlevel (altogether 5 levels), each 10 μm thick (Leica CM 3050S) weresagittally cut. Brain levels were chosen according to the morphologyatlas “The Mouse Brain” from Paxinos and Franklin (2nd edition). The cutof the five levels started with a random slice then sampling continueduniformly and systematically, always retaining 15 slices per level inseries and discarding 150 μm in between the levels. For determination ofTAU pathology in the hippocampus and the amygdala 5 slices (1 from eachlevel) per brain region and animal were stained using AT180 (# MN1040,Thermo Scientific) and HT7 (# MN1000, Thermo Scientific) antibodies. Theprimary antibodies were visualized by Cy-3-coupled secondary antibody(Jackson Laboratories) and subsequently immunoreactive area wereevaluated using Image Pro Plus (v6.2) software.

Immunoreactive objects were measured above a size restriction (30 μm² inthe amygdala, 7 μm² in the hippocampus) and above a dynamic intensitythreshold. Total area and intensity of objects and the individualthreshold were automatically filed. If used, a dynamic threshold wasdefined as “mean intensity within AOI plus factor times the standarddeviation of pixel intensities within the AOI”. In any case, values hadto exceed a minimal set threshold. Exact threshold levels are given inthe table below.

Thresholds Minimum Dynamice factor AT180 Amygdala 25 2 AT180 28 —Hippocampus HT7 Amygdala 35 2 HT7 Hippocampus 25 0.5

The region size was measured by manual delineation of the hippocampusand amygdala. HT7 and AT180 IR area data were normalized to the regionssize.

All IHC related data with n>4 followed a Gaussian distribution accordingto Kolmogorov Smirnov normality test and are represented as mean+SEM.For the vehicle group consisting of four animals only, thus too few fornormality testing, Gaussian distribution was assumed. Group differenceswere calculated by means of a parametric one-way ANOVA followed byNewman Keuls post hoc testing, calculated with GraphPadPrism software.The alpha-error level was set to 0.05.

Brain TAU pathology was determined in hippocampus and amygdala byimmunohistochemical (IHC) quantitation using AT180 (anti-pTAU) and HT7(anti-TAU) antibodies. Furthermore, the treatment effects on solublepTAU and TAU in cortex and hippocampus was measured in the solublehomogenate fraction using MesoScale Discovery (MSD) duplex technology,probing for pTAU and total TAU.

None of the antibodies used for either the IHC or the MSD assays have anepitope that overlaps with the two treatment antibodies used in thisstudy.

6.1.5. Generation of Fraction for the Quantification of Soluble TauProtein Level in the Soluble Brain Fractions of Tg Mice

Mice treated according to method 6.1.1. were euthanized 1 day followingthe second administration to determine Tau pathology in the brain.Briefly, soluble cortex samples from one brain hemisphere werehomogenized in 100 to 200 μL of cold extraction buffer (25 mM Tris-HClpH=7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM β-glycerophosphate, 30mM NaF, 2 mM Na₃VO₄, protease and phosphatase inhibitor cocktail). Thehomogenates were centrifuged (74,200×g for 15 min at 4° C.) and thesupernatants were used for the analysis of soluble Tau. Theconcentration of total protein in the soluble fractions of cortexsamples was determined by a BCA protein quantitation assay (ThermoFisher Scientific, Rockford, Ill., USA).

6.1.6. Analysis of pTau Presence by Western Blot

To probe for immunoreactivity in the brains of mice administeredACI-36-2B6-Ab2 and ACI-36-3A8-Ab2, two antibodies reported to bind pTauPHF epitopes (Greenberg et al., 1992; Reig et al., 1995; Hoffmann etal., 1997) were used in Western-blot (WB) assays. Soluble fractions fromcortex were diluted by adding an equal volume of sample buffer A (125 mMTris-HCl pH 6.8, 4% [w/v] sodium dodecyl sulfate [SDS], 20% glycerol,0.01% bromophenol blue, 5% β-mercaptoethanol), and the samples wereheated to 95° C. for 10 min. 30 μg of sample was loaded onto a 4-12%Bis-Tris gel (Invitrogen, Basel, Switzerland) and run in MOPS SDS buffer(Invitrogen). Proteins were transferred to a 0.45 μm PVDF membrane intransfer buffer (25 mM Tris pH 8.6, 190 mM glycine, 20% methanol). Toverify protein transfer, the membranes were stained with Ponceau S for 5min, washed, and blocked for 1 hour in blocking buffer (5% BSA in TBS[50 mM Tris-HCl, pH 7.6, 150 mM NaCl]). Membranes were blottedover-night at 4° C. with the primary antibodies in blocking buffer and0.1% Tween. The two pTau PHF-specific primary antibodies used for theWBs were: anti-pS396 (PHF-13 epitope; AbCam, Cambridge, UK; used at 3μg/mL), specific to phosphorylated Ser396 (pS396) of human or murinepTau (Hoffmann et al., 1997), and AD2 (PHF-1 epitope; BioRad, Reinach,Switzerland; used at 0.4 μg/mL), specific for human and murine pS396 andphosphorylated Ser404 (pS404; Reig et al., 1995). For total Tau WBs,Tau5 (0.5 μg/mL), an antibody that binds both human and murine Tau (BDBiosciences, Allschwil, Switzerland), was used. Following incubationwith the primary antibody, membranes were washed with 0.1% Tween in TBS,and incubated with the secondary antibodies: goat anti-mouse-IRDye800 orgoat anti-rabbit-IRDye680 (both from Li-Cor Biosciences, NE, USA), bothdiluted 1:15000 in BB and 0.1% Tween. Membranes were then incubated 1hour at room-temperature protected from light, washed for 15 min 3-timeswith 0.1% Tween in TBS, and for 5 min 2-times with TBS, and bandsquantified using Li-Cor Odyssey near-infrared imaging system (Li-Cor).Bands were normalized to β-actin expression (AbCam; used at 0.4 μg/mL).To verify the identification of the human transgenic versus the mouseendogenous Tau bands, blots were probed with an antibody specific forhuman total Tau (Tau13, AbCam; not shown). Additionally, membranes wereprobed with an anti-mouse primary antibody, to verify that the treatmentantibodies, ACI-36-2B6-Ab2 and ACI-36-3A8-Ab2, were not present in thedenatured test samples in sufficient quantity to interfere with thebinding of anti-pS396 or AD2. No intact or denatured treatmentantibodies were detected (results not shown) in the samples used forthis study.

6.1.7. Statistical Analysis

Data were analyzed using non-parametric Kruskal-Wallis rank sumstatistics, and if significant at the P<0.05 level, a Dunn's post-hoctest was used comparing all groups (GraphPad Prism, GraphPad Software,CA, USA). Results are presented as individual data points showingmean±SEM. Differences with P<0.05 were considered as statisticallysignificant.

6.2 Results 6.2.1. Brain TAU Pathology by Immunohistochemical (IHC)Quantitation

Two i.p. injections of ACI-36-2B6-Ab2 and ACI-36-3A8-Ab2 did not showany gross adverse effects during the study period. Staining for pT231and pS235 using AT180 by IHC, showed increased immunoreactive area (IR)in the amygdala following ACI-36-3A8-Ab2 treatment ( ). Mice treatedwith 3 mg/kg ACI-36-2B6-Ab2 had significantly less AT180 IR area in thehippocampus ( ).

ACI-36-3A8-AB2 treatment increased AT180 IR pTAU compared to the PBSgroup in the amygdala. In the hippocampus ACI-36-2B6-AB2 treatmentdecreased pTAU. AT180 specifically labels pTAU. The frequency of AT180IR cells was decreased in ACI-36-2B6-AB2 treated mice. This effect wasstronger in the low dose (3 mg/kg) group (ACI-36-2B6-AB2 LD). The somalstaining pattern does not differ among groups.

At the higher 10 mg/kg dose, a non-significant trend for less AT180 IRwas seen in the hippocampus for both ACI-36-2B6-Ab2 and ACI-36-3A8-Ab2,when compared to vehicle control treated mice. Qualitatively,ACI-36-2B6-Ab2 treated animals showed a lower number of hippocampalneurons with highly intense AT180 labeling.

6.2.2. Reduction of Total Tau Level in Brain Fraction Following PassiveImmunization

The effect of the treatments on pTAU and TAU in the brain fractioncontaining soluble proteins was measured using an MSD duplex assay.Levels of total soluble TAU in the cortex was significantly reduced inmice treated with ACI-36-2B6-Ab2 and ACI-36-3A8-Ab2 (p<0.01; FIG. 3upper panel). The levels of soluble pTAU was also significantly reduced(p<0.05; FIG. 3 lower panel), with the 3 mg/kg dose of ACI-36-2B6-Ab2demonstrating the greatest decrease (p<0.01). The ratio of pTAU to totalTAU remained unchanged. The levels of soluble TAU and pTAU did notchange in samples from hippocampus (not shown here).

6.2.3. Effects of ACI-36-2B6-Ab2 and ACI-36-3A8-Ab2 Administration onthe Presence of Phospho-Tau Epitopes Present in Paired Helical Filaments(PHFs)

Structurally, neurofibrillary tangles (NFTs) consist of paired helicalfilaments (PHFs) composed of the microtubule-associated protein Tau,found primarily in a hyper-phosphorylated state (Alonso et al., 1997).The objective of this study was to use antibodies that recognize pTauPHF to probe for and quantify these pTau PHF epitopes in the brains ofTau transgenic mice, following the administration of ACI-36-2B6-Ab2 andACI-36-3A8-Ab2.

To measure the effects of two ACI-36-2B6-Ab2 or ACI-36-3A8-Ab2administrations on the quantity of well documented Tau PHF phosphoepitopes, brain cortex soluble fractions from treated Tau Tg mice wereprobed with AD2 (PHF-1 epitope, pS396/pS404) and anti-pS396 antibody(PHF-13 epitope, pS396) using WBs. The immunoreactivity was quantifiedusing an infrared imaging system. The effects of ACI-36-3A8-AB2 andACI-36-2B6-AB2 treatment on AD2 PHF immunoreactivity in the cortex ofTau Tg mice were determined using AD2 which probes for pS396 and pS404,two previously documented PHF phospho residues of Tau (Greenberg et al.,1992; Reig et al., 1995).

Bands, indicating human and mouse pTau phosphorylated on S396 and S404using the AD2 (PHF-1) antibody, were quantified using a Li-Cor infraredimaging system. Values for individual mice as well as the mean±SEM aredetermined.

A non-significant trend was observed for a reduction in AD-2-positivepTau immunoreactivity was observed for the transgenic human pTau band.However, a significant reduction in the quantity of mouse AD2-positivepTau was observed in mice treated with 3 mg/kg of ACI-36-2B6-Ab2, and anon-significant trend when treated with either 10 mg/kg ofACI-36-2B6-Ab2 or ACI-36-3A8-Ab2.

When a different antibody that specifically recognizes pTau pS396 wasused for staining (Hoffmann et al., 1997), an even greater effect wasobserved. Mice treated with 3 mg/kg of ACI-36-2B6-Ab2 had significantlyless pS396-positive human transgenic and mouse endogenous pTau, with atrend towards reduction when treated with 10 mg/kg ACI-36-2B6-Ab2 orACI-36-3A8-Ab2. To assess the effects on total human and mouse Tau,which includes both non-phosphorylated and all pTau, blots were probedwith the Tau5 antibody. Compared to vehicle control, total Tau was notmodulated by ACI-36-2B6-Ab2 or ACI-36-3A8-Ab2 administered at 10 mg/kg,however a trend for reduced total Tau was observed for mice administeredACI-36-2B6-Ab2 at 3 mg/kg.

6.2.4 Summary

Two peripheral administrations of Tau Tg mice with the anti-pTAUantibody ACI-36-3A8-Ab2 significantly reduced soluble TAU and solublepTAU in the brain cortex. Two peripheral administrations of Tau Tg micewith the anti-pTAU antibody ACI-36-2B6-Ab2 significantly reduced solubleTAU and soluble pTAU in the brain cortex. Additionally, ACI-36-2B6-Ab2significantly reduced pTAU immunoreactivity in the hippocampus. Theseresults demonstrate the ability of passive anti-pTAU immunization, usingACI-36-2B6-Ab2 and ACI-36-3A8-Ab2 antibodies, in reducing tauopathy.

Two peripheral administrations of ACI-36-2B6-Ab2 at 3 mg/kg to Tau Tgmice reduced the presence of pTau PHF epitopes in the cortex as measuredby Western-blotting. At a higher dose of 10 mg/kg, both ACI-36-2B6-Ab2and ACI-36-3A8-Ab2 showed a trend towards reduced pTau PHF epitopeimmunoreactivity. These results show that ACI-36-2B6-Ab2 andACI-36-3A8-Ab2 antibodies may be suitably used in passive immunotherapyagainst tauopathies such as Alzheimer's Disease.

Example 7 1-Month Treatment of Human Tau Over-Expressing Mice 7.1Methods 7.1.1 Mice and Treatments

Tau transgenic mice were used and administered the treatment antibodiesas shown in the Table in Method 6.1. (study no. 2.)

7.1.2. Behavioral Testing—Morris Water-Maze (MWM) Task

Following the last administration, a water-maze (MWM) task was performedto test for spatial memory performance on mice treated according to6.1.1. The MWM testing was performed with all enclosed animals in week 4after start. The MWM consists of a white circular pool with a diameterof 100 cm, filled with tap water at a temperature of 21±2° C. The poolis virtually divided into four sectors. A transparent platform (8 cmdiameter) is placed about 0.5 cm beneath the water surface. During alltest sessions, the platform is located in the southwest quadrant of thepool. Each mouse had to perform three trials on each of four consecutivedays. A single trial lasted for a maximum of one minute. During thistime, the mouse had the chance to find the hidden, diaphanous target.After each trial mice were allowed to rest on the platform for 10-15 secto orientate in the surrounding. At least one hour after the last trialon day 4, mice had to fulfill a so-called probe trial (PT). During thePT, the platform was removed from the pool and the number of crossingsover the former target position was recorded by the experimentertogether with the abidance in this quadrant. For the quantification ofescape latency (the time [seconds] the mouse needed to find the hiddenplatform and therefore to escape from the water), of pathway (the lengthof the trajectory [meter] to reach the target), of target zone crossingsand of the abidance in the target quadrant in the PT, a computerizedtracking system (Biobserve Software) was used. All animals had toperform a visual test after the PT on the last day to exclude influenceof insufficient seeing abilities on behavioral results.

7.1.3. Brain Tau Pathology Determination by Immunohistochemical (IHC)Quantitation

Mice were euthanized 1 day following the MWM (1 week following lastadministration) to determine Tau pathology in the brain. Brain Taupathology was determined in hippocampus and amygdala byimmunohistochemical (IHC) quantitation using AT180 (anti-pTau,pT231/pS235) and HT7 (human-specific anti-Tau) antibodies. Furthermore,the treatment effects on soluble pTau and soluble Tau in cortex andhippocampus was measured in the homogenate fraction using MesoScaleDiscovery (MSD) duplex technology, probing for pTau (pT231) and totalTau. None of the antibodies used for either the IHC or the MSD assayshave an epitope that overlaps with the treatment antibody used in thisstudy.

7.1.4. Sample Preparation for the Analysis of Soluble Tau in Cortex andHippocampus

Mice were euthanized for tissue collection, one week following the lasttreatment administration. Cortex and hippocampus were homogenized in 100to 200 μL of cold extraction buffer 1 (25 mM Tris HCl pH=7.4, 150 mMNaCl, 1 mM EDTA, 1 mM EGTA, 10 mM β-glycerophosphate, 30 mM NaF, 2 mMNa₃VO₄, protease and phosphatase inhibitor cocktails). The homogenateswere centrifuged (74,200 g for 15 min at 4° C.) and the supernatantswere used for the analysis of soluble Tau in cortex and hippocampus(FIG. 4A). The pellets were resuspended in 100-200 μL extraction buffer2 (10 mM Tris HCl pH=7.4, 800 mM NaCl, 300 mM sucrose, 1 mM EGTA,protease and phosphatase inhibitor cocktails) and transferred to a 1.5mL tube. The solutions were centrifuged (4,000 g for 20 min at 4° C.)and the supernatants transferred to ultracentrifugation tubes. Sarkosyl(a 30% aqueous solution) was then added to a final concentration of 1%and incubated for 1.5 hours at room temperature. After centrifugation(74,200 g for 30 min at 4° C.) the supernatants were discarded and thepellets were re-suspended in 100 μL buffer 3 (50 mM Tris-HCl, pH=7.4).The re-suspended pellets were used as sarkosyl-insoluble (Sin T) Tau incortex and hippocampus. The concentration of total protein in thesoluble and Sin T fractions samples was determined by a BCA proteinquantitation assay (Thermo Fisher Scientific, Rockford, Ill., USA).

7.1.5. Western Blots for pTau PHF and Tau

To evaluate the effect of ACI-36-2B6-Ab1 administration on the presenceof pTau PHF in brain cortex and hippocampus, two antibodies reported tobind pTau PHF epitopes (Greenberg et al., 1992; Reig et al., 1995;Hoffmann et al., 1997) were used in Western-blot (WB) assays. Solubleand Sin T fractions from cortex and hippocampus were diluted by addingan equal volume of sample buffer A (125 mM Tris-HCl pH 6.8, 4% [w/v]sodium dodecyl sulfate [SDS], 20% glycerol, 0.01% bromophenol blue, 5%β-mercaptoethanol), and the samples were heated to 95° C. for 10 min. 30μg of sample was loaded onto a 4-12% Bis-Tris gel (Invitrogen, Basel,Switzerland) and run in MOPS SDS buffer (Invitrogen). Proteins weretransferred to a 0.45 μm PVDF membrane in transfer buffer (25 mM Tris pH8.6, 190 mM glycine, 20% methanol). To verify protein transfer,membranes were stained with Ponceau S for 5 min. Membranes were thenwashed, and blocked for 1 hour in blocking buffer (5% BSA in TBS [50 mMTris-HCl, pH 7.6, 150 mM NaCl]). Membranes were blotted over-night at 4°C. with the primary antibodies in blocking buffer and 0.1% Tween.

The two pTau PHF-specific primary antibodies used for the WBs were:anti-pS396 (PHF-13 epitope; AbCam, Cambridge, UK; used at 3 μg/mL),specific to phosphorylated Ser396 (pS396) of human or murine pTau(Hoffmann et al., 1997), and AD2 (PHF-1 epitope; BioRad, Reinach,Switzerland; used at 0.4 μg/mL), specific for human and murine pS396 andphosphorylated Ser404 (pS404; Reig et al., 1995). For detection oftarget effects, ACI-36-2B6-Ab1 was used for blotting at 1.6 μg/mL. Fortotal Tau WBs, Tau5, an antibody that binds both human and murine Tau(BD Biosciences, Allschwil, Switzerland), was used at 0.5 μg/mL. Allmembranes were additionally blotted for β-actin (AbCam; used at 0.4μg/mL) to normalize for protein loading.

Following incubation with the primary antibody, membranes were washedwith 0.1% Tween in TBS, and incubated with the secondary antibodies:goat anti-mouse-IRDye800 or goat anti-rabbit-IRDye680 (both from Li-CorBiosciences, NE, USA), both diluted 1:15,000 in BB and 0.1% Tween.Membranes were then incubated 1 hour at room-temperature protected fromlight, washed for 15 min 3-times with 0.1% Tween in TBS, and for 5 min2-times with TBS, and bands quantified using Li-Cor Odysseynear-infrared imaging system (Li-Cor). Bands of interest were normalizedto β-actin expression. To verify the identification of the humantransgenic versus the mouse endogenous Tau bands, blots were probed withan antibody specific for human total Tau and does not cross-react withmurine Tau (Tau13, AbCam; not shown). Additionally, membranes wereprobed with an anti-mouse primary antibody, to verify that thetreatment-antibody, was not present in the denatured test samples insufficient quantity to interfere with the binding of the primaryblotting antibodies. No intact or denatured treatment antibodies weredetected (results not shown) in the samples used for this study. Valuesare expressed as arbitrary β-actin-corrected immunoreactivity (IR).

7.1.6. Statistical Analysis

Data were analyzed using a one-way ANOVA, followed by Dunnett's multiplecomparison post-hoc test (GraphPad Prism, GraphPad Software, CA, USA)comparing each treatment to Tg control-treated mice. Results arepresented as individual data points showing mean±SEM. Differences withp<0.05 were considered as statistically significant. Single values thatwere identified as significant (p<0.05) outliers by Grubb's extremestudentized deviate test, were excluded

7.2 Results 7.2.1. Behavioral Testing—Morris Water-Maze (MWM) TaskFollowing Passive Immunization

Four i.p. injections of ACI-36-2B6-Ab1 administered weekly at 3 mg/kg or1 mg/kg over a four week period did not show any gross adverse effects.

During the last week of treatment, spatial navigation learning andmemory of animals were evaluated. Animals had to fulfill 4 days oftraining with 3 trials per day followed by one probe trial and visualtest. Escape latency (the time [seconds] the mouse needed to find thehidden platform and therefore to escape from the water), the pathway(the length of the trajectory [meter] to reach the target), the swimspeed (calculated quotient of pathway and escape latency), the number oftarget crossings and the abidance in the target quadrant were evaluated.

Tg (group A) as well as nTg (F) control animals treated with vehicleshowed the expected learning curves when evaluating escape latency andlength of the swimming path to reach the platform over the four testingdays. Tg control (A) animals had a significant learning impairment asshown by flatter learning curves in escape latencies and swimming pathscompared to nTg control animals (F). Escape latencies and swimming pathwere significantly (Two Way ANOVA) longer on training days 3 and 4(p<0.001; Bonferroni's post test). Treatment with ACI-36-2B6-Ab1 andACI-36-3A8-Ab1, low or high dose (groups B and C and D and E,respectively) did not lead to a significant improvement of spatiallearning abilities compared to Tg control animals (group A) and showedsimilar learning curves. When adjusting the day 1 performance of eachgroup to 100% and all further days as percentage of day 1, animprovement can be seen for the ACI-36-3A8-Ab1 treated mice (bothdosages). The effect reached statistical significance for swimming pathlength on day 3 (p<0.01 group D and p<0.05 group E) and day 4 (p<0.05group D).

For the ACI-36-2B6-Ab1 treated mice (both dosages) a slight improvementcan be seen in swimming path length, although without statisticalsignificance.

No differences between treatment groups were detected in terms ofswimming speed on all four training days.

The results from the MWM test demonstrated trends toward improvedspatial learning for mice treated with ACI-36-2B6-Ab1 andACI-36-3A8-Ab1.

7.2.2. Brain TAU Pathology by Immunohistochemical (IHC) Quantification

The AT180 antibody stains the endogenous and human pTAU (doublyphosphorylated at Thr231 and Ser235).

AT180 IR in the amygdala and hippocampus after ACI-36-2B6-AB1 andACI-36-3A8-AB1 immunization was determined. The AT180 IR area percentagein the amygdala and hippocampus was measured.

The amount of intrasomal pTAU in nTg controls was significantly lowercompared to Tg groups (p<0.001). In the amygdala, a tendency to increasesomal pTAU was observed for the 3 mg/kg ACI-36-2B6-Ab1 treatment. Incontrast, both dosages of ACI-36-2B6-Ab1 tended to lower pTAU comparedto vehicle treated animals in the hippocampus. Mean staining intensitieswere comparable in all transgenic groups.

ACI-36-3A8-Ab1 treatment did not alter somal pTAU in the hippocampus andamygdala and neuronal pTAU levels in the amygdala and hippocampus didnot differ significantly among treated transgenic groups. Mean and sumof staining intensities were comparable in all transgenic groups.

Since the HT7 antibody is specific for human TAU, only little signal wasmeasured in nTg controls, that derives from autofluorescence oflipofuscine dots above seven pixel in size. ACI-36-2B6-Ab1 treatment didnot alter somal HT7 positive IR area in the hippocampus ( ) compared tothe vehicle control (PBS). In the amygdala, mice receiving the lowerdose of ACI-36-2B6-Ab1 tended to have higher levels of total human TAU(T-test: p=0.0954) in terms of IR area ( ). This increase was alsoqualitatively visible as increase of the area of staining and thestaining intensity in individual neuronal somata. No statisticallysignificant treatment induced differences were observed in thehippocampal neurons.

ACI-36-3A8-Ab1 treatment did not significantly alter somal HT7 positiveIR area in the amygdala ( ) and the hippocampus ( ) compared to thevehicle control (PBS). Mean and sum of staining intensities werecomparable in all transgenic groups (data not shown).

Brain Tau pathology did not show a change in total Tau or pTau levels inthe brain soluble fraction, however immunostaining of brain sectionsdemonstrated a reduction in hippocampus pTau in mice treated withACI-36-2B6-Ab1.

7.2.3. Effects of Anti-Tau Antibody Administration on Phospho-TauEpitopes Present in Paired Helical Filaments (PHFs)

Alzheimer's Disease (AD) is characterized neuropathologically byneurofibrillary tangles (NFTs; Braak, Braak, & Bohl, 1993).Structurally, NFTs consist of paired helical filaments (PHFs) composedof the microtubule-associated protein Tau, found primarily in ahyper-phosphorylated state (Alonso et al., 1997). The objective of thisstudy was to reduce these pTau PHF epitopes in the brains of Tautransgenic mice by four administrations of the anti-pTau antibodyACI-36-2B6-Ab1 and ACI-36-3A8-Ab1.

To measure the effects of four ACI-36-2B6-Ab1 administrations on thequantity of well documented Tau PHF phospho epitopes, brain cortex andhippocampus soluble and Sin T fractions from treated Tau Tg mice wereprobed with AD2 (PHF-1 epitope, pS396/pS404) and anti-pS396 antibody(PHF-13 epitope, pS396) using WBs. As markers of Tau PHFs, the presenceof pS396/pS404 have been previously documented (Greenberg et al., 1992;Reig et al., 1995), and more specifically the pS396 site (Hoffmann etal., 1997). In the Tau Tg mice, Tau is expressed as endogenous murineTau and as the human Tau transgene, with a molecular-weight differencethat can be clearly identified on WBs when a blotting antibody binds Taufrom both species and Tau from the two different transcripts isexpressed in sufficient amounts. Therefore when possible, the endogenousmouse and human transgenic Tau bands were identified for each blottingantibody and quantified separately. To verify the migration patterns forthese Tau bands in our WB assays, an anti-Tau antibody that binds tototal Tau, but is human specific (Tau13), and therefore only shows thehuman transgene in Tau Tg brains was used for Tau Tg control samples toverify migration patterns for human and mouse Tau in the Tau transgenicmice. Additionally, all quantified bands were normalized to β-actin.

The presence of PHF epitopes, probed for in the soluble fraction frombrain cortex, was reduced in ACI-36-2B6-Ab1 and in ACI-36-3A8-Ab1treated mice. This was significant for both the mouse and human bands,using the pS396 (PHF-13) antibody for the WBs (FIGS. 5A and B).

When the AD2 (PHF-1, pS396/pS404) antibody was used for WBs of extractsfrom ACI-36-2B6-Ab1 and ACI-36-3A8-Ab1 treated mice, a significantreduction was observed (FIG. 5C).

A note should be made that even though the two PHF-specific antibodiesthat were used for these WBs have similar epitopes and good specificityto their phosphorylated target(s), the pS396 (PHF-13) antibody appearsto have a better signal-to-noise ratio and was the better overallantibody for these WBs.

The direct target effect of the treatment-antibody was probed for in thecortex using ACI-36-2B6-Ab1 and the ACI-36-2B6-Ab1, respectively, as theblotting antibody. This anti-pTau antibody binds to the same phospho-Tauepitope as the treatment antibody used in this study. Blots hadpreviously been probed with a secondary anti-mouse IgG antibody only.

Bands were quantified using an infrared imaging system. Values forindividual mice as well as the mean±SEM are determined.

No signal above background was detected, verifying the lack of blockingeffects or interference by the treatment antibody in these samples (datanot shown).

In ACI-36-2B6-Ab1 treated mice, a trend towards a reduced signal down tothe level of control-treated nTg mice was observed, indicative of adirect target effect (FIG. 5G). In ACI-36-3A8-Ab1 treated mice nosignificant effects of treatment were observed (FIG. 5G).

The significant effect of ACI-36-2B6-Ab1 treatment on total Tau in thesoluble cortex of Tau Tg mice was observed, using a Tau5 antibody forblotting which binds both the endogenous mouse Tau and the transgenichuman Tau (FIGS. 5H and 5I). A significant reduction in total Tau wasobserved for both endogenous mouse Tau and the transgenic human Tau inthe soluble fraction of brain cortex.

Bands, indicating the mouse A) and human B) total Tau (Tau5), werequantified using an infrared imaging system. Values for individual miceas well as the mean±SEM are determined.

The presence of PHF epitopes in the detergent insoluble Tau fraction wasdone by preparing sarkosyl-insoluble (Sin T) brain fractions.

Bands were quantified using an infrared imaging system. Values forindividual mice as well as the mean±SEM are determined.

Much less Tau was present in this fraction when compared to the solubleTau fraction, both in the cortex and in hippocampus. This may be due tothe age of the Tau Tg mice used in this study, which at 4 months may nothave accumulated a significant amount of insoluble and aggregated Tau inthe hippocampus and cortex. Therefore, when probing for PHF epitopes inthe Sin T fraction, only the AD2 (PHF-1, pS396/pS404) antibody provideda signal that was sufficient for reliable quantization of bands.

Mice treated with 1 mg/kg of ACI-36-2B6-Ab1 and with 1 mg/kg ofACI-36-3A8-Ab1, respectively, had a significant reduction in the PHF-1epitope in bands representing the endogenous mouse Tau (FIG. 5C).Signals observed for the transgenic human band were not intense enoughto be quantitated reliably.

The hippocampus was also probed, using the same antibodies and fractionsas that for the cortex. Lower signals for all blotting antibodies weredetected in fractions from hippocampus compared to that of cortex.

The effects of ACI-36-2B6-AB1 and ACI-36-3A8-AB1 treatment on pS396(PHF-13) immunoreactivity in the soluble hippocampus of Tau Tg mice wasdetermined. Bands, indicating the mouse A) and human B) pTau pS396(PHF-13) epitopes, were quantified using an infrared imaging system.Values for individual mice as well as the mean±SEM are determined.

ACI-36-2B6-Ab1 treatment did not significantly alter the presence of thepS396 (PHF-13) epitope in the mouse Tau soluble hippocampus fraction,with a small trend for a reduction in the human transgene band.

ACI-36-3A8-Ab1 treatment showed trends towards a reduction in thepresence of the pS396 (PHF-13) epitope in the mouse Tau solublehippocampus fraction and the human transgene band.

Similarly to what was observed for pS396 (PHF-13) WBs, a trend for areduced signal was detected in both extracts of ACI-36-2B6-Ab1 andACI-36-3A8-Ab1 treated mice for total Tau in the human Tau solublehippocampus fraction.

Akin to the cortex Sin T samples, the Sin T fraction from hippocampushad very low levels of pTau. Mice treated with ACI-36-2B6-Ab1 andACI-36-3A8-Ab1, respectively, had no change in the PHF-1 (pS396/pS404)epitope in bands representing the endogenous mouse Tau.

Signals observed for the transgenic human band were not intense enoughfor reliable quantization.

7.2.4 Summary

The study indicates that passive immunization using four administrationsof a phosphosite-specific anti-pTau antibody ACI-36-2B6-Ab1 andACI-36-3A8-Ab1 antibodies improves spatial learning and reduces brainpTau pathology.

Four peripheral administrations of the anti-pTau antibody ACI-36-2B6-Ab1at 1 and 3 mg/kg to Tau Tg mice reduced the presence of pTau PHFepitopes in the cortex as measured by Western-blotting. A trend forreduction was observed in the hippocampus. Similarly, a reduction intotal Tau was also observed. A significant reduction in pTau PHF-1immunoreactivity was observed in the insoluble cortex fraction, and atrend was also observed which indicated direct target effects of theantibody treatment. These results provide further support for anti-pTauantibodies ACI-36-2B6-Ab1 and ACI-36-3A8-Ab1 in passive immunotherapyagainst tauopathies such as Alzheimer's Disease.

Example 8 3-Month Treatment of Human Tau Over-Expressing Mice 8.1Methods 8.1.1 Mice and Treatments

Tau transgenic mice were used and administered the treatment antibodiesas shown in the Table in Method 6.1. (study no. 3) and mice wereassigned to 4 different treatment groups as described in the tablebelow.

Mouse Geno Group strain type Age at start Sex n Treatment A TMHT Tg 3months mixed 15 + 1 PBS i.p. 10 μl/g weekly (±2 weeks) (control) b.w. BTMHT Tg 3 months mixed 15 + 1 ACI-36- i.p. 10 μl/g weekly (±2 weeks)2B6-Ab1 b.w. (1 mg/kg) C TMHT Tg 3 months mixed 15 + 1 ACI-36- i.p. 10μl/g weekly (±2 weeks) 2B6-Ab1 b.w. (3 mg/kg) F TMHT nTg 3 months mixed15 + 1 PBS i.p. 10 μl/g weekly (±2 weeks) (control) b.w.

In total 45 Tg mice plus 3 reserves allocated to treatment groups A to Cand 15 nTg mice plus 1 reserve (group F) were treated on day 0, 7, 14,21, 28, 35, 42, 49, 56, 63, 70, 77, and 84 by i.p. injection of eitherPBS (vehicle control) or anti-pTAU antibody, ACI-36-2B6-Ab1 orACI-36-3A8-Ab1. Animals were randomly enclosed in 5 different startinggroups (scales) comprising animals of all treatment groups. The numberof animals in a scale was limited to ensure same age and uniformhandling. Following the 12^(th) administration, a water-maze (MWM) taskwas performed to test for spatial memory performance. Following the MWM,mice were administered the test article one additional time (13^(th)injection) before being euthanized 24 hours later do determine Taupathology. Brain Tau pathology was determined in hippocampus andamygdala by immunohistochemical (IHC) quantitation using the AT180(anti-pTau, pT231/pS235) antibody. Furthermore, the treatment effects onsoluble and sarkosyl-insoluble Tau and pTau in cortex and hippocampuswere measured using MesoScale Discovery (MSD) technology, probing forpTau (pT231 and pS396) and total Tau.

8.1.2. Behavioral Testing—Morris Water-Maze (MWM) Task

This experiment was performed according to the protocol described inExample 7.1.2. In week 12, spatial navigation was tested in the MorrisWater Maze (MWM) to evaluate learning and memory.

8.1.3. Molecular Biology

Total TAU and Tau phosphorylated at Thr231 and at pS396 was quantifiedin brain homogenates of Tg animals by using an immunosorbent assay fromMesoScale Discovery (MSD)

8.1.4. Brain Tau Pathology Determination by Immunohistochemical (IHC)Quantitation

This experiment was performed according to the protocol described inExample 7.1.3. TAU pathology was determined by AT180 immunoreactivity inthe hippocampus and amygdala of 8 animals per group.

8.1.5. Effects of Three Month Anti-Tau Antibody Administration onPhospho-Tau Epitopes Present in Paired Helical Filaments (PHFs)

This experiment was performed according to the protocols described in7.1.4., 7.1.5. and 7.1.6. To measure the effects of four ACI-36-2B6-Ab1and ACI-36-3A8-Ab1 administrations on the quantity of well documentedTau PHF phospho epitopes, brain cortex and hippocampus soluble and Sin Tfractions from treated Tau Tg mice were probed with AD2 (PHF-1 epitope,pS396/pS404), anti-pS396 antibody (PHF-13 epitope, pS396) and AT180(pT231/pS235) using WBs.

8.1.6. Effect of 3 Month Anti-Tau Antibody Administration on Phospho-TauEpitopes Using biGT Tau Bigenic Mice

Study no. 4 was done using bigenic Tau mice as shown in Method 6.1.Brain cortex samples were prepared as shown in the figure below, usingthe total homogenate (TH) or the soluble fraction (51) for westernblotting. Membranes were probed using the following blotting antibodiesfor pTau or total Tau:

-   -   HT-7 (26 ng/ml), specific to total human Tau    -   PHF-13 (pS396) at 1/7500 dilution    -   AT180 (pT231) at 2.47 ug/ml    -   AT8 (pS202) at 3 ug/ml    -   pS404 at 1:5000 dilution    -   pS400 at 1:5000 dilution

All quantifications were normalized to β-actin.

8.2 Results for ACI-36-2B6-Ab1 Antibody

Thirteen i.p. injections of ACI-36-2B6-Ab1 administered weekly at 1 or 3mg/kg over a twelve week study period did not show any gross adverseeffects.

8.2.1 Behavioral Results—Morris Water Maze

The results from the MWM test demonstrated strong trends toward improvedspatial learning for mice treated with ACI-36-2B6-Ab1 (FIG. 9).

During the last week of treatment, spatial navigation learning andmemory of animals were evaluated. Animals had to fulfill 4 days oftraining with 3 trials per day followed by one probe trial and visualtest. Escape latency (the time [seconds] the mouse needed to find thehidden platform and therefore to escape from the water), the pathway(the length of the trajectory [meter] to reach the target), the swimspeed (calculated quotient of pathway and escape latency), the number oftarget crossings and the abidance in the target quadrant were evaluated.Vehicle treated Tg (group A) and nTg (group F) control animals showedexpected learning curves in terms of escape latency and length of theswimming path to reach the platform over the four testing days. Tgcontrol (A) animals showed a significant impairment of learningabilities reflected in flatter learning curves of escape latencies andswimming paths compared to nTg control animals (group F). Escapelatencies and swimming paths were significantly (Two Way ANOVA)different on training days 3 (p<0.01, latency; p<0.001, length;Bonferroni's post test) and 4 (p<0.01; Bonferroni's post test).Treatment with ACI-36-2B6-Ab1, low or high dose (B and C) did notsignificantly improve spatial learning abilities compared to Tg controlanimals (A). When adjusting the performance of each group to 100% ontraining day 1 and all further days as percentage of day 1, a slightimprovement can be seen for the ACI-36-2B6-Ab1 treated mice (low andhigh dosage) in swimming path length, although without statisticalsignificance. No differences between treatment groups were detected whencalculating the swimming speed on all four training days. In the probetrial (PT), the abidance in the target quadrant (south west quadrant) aswell as target zone crossings were recorded. nTg controls (group F)spent more time in the target quadrant and crossed the target zone moreoften relative to Tg controls (group A) but without statisticalsignificance.

Treatment with neither the low nor the high dose led to an improvementof spatial learning abilities in comparison to Tg control mice asevaluated in the PT.

8.2.2 Molecular Biology 8.2.2.1 TAU in Soluble Fraction of CortexHomogenates

Total TAU, p231TAU, p396TAU, and the ratios of pTAU to total TAU wereevaluated in the soluble fraction of cortex homogenates of n=16 animalsfrom group A (Tg vehicle group; PBS), B (Tg, ACI-36-2B6-Ab1 1 mg/kg),and C (Tg, ACI-36-2B6-Ab1 3 mg/kg). A treatment with ACI-36-2B6-Ab1 didnot significantly affect total TAU and pTAU in the in soluble cortexhomogenates. However, a slight increase (without significance) of meantotal TAU, p231TAU, and p396TAU was observed upon ACI-36-2B6-Ab1treatment. TAU phosphorylation evaluated as the ratios of pTAU to totalTAU was not affected.

8.2.2.2 TAU in Sarcosyl Insoluble Fraction of Cortex Homogenates

Total TAU, p231TAU, p396TAU, and the ratios of pTAU to total TAU wereevaluated in the sarcosyl insoluble fractions of cortex homogenates ofn=16 animals from group A (Tg vehicle group; PBS), B (Tg, ACI-36-2B6-Ab11 mg/kg), and C (Tg, ACI-36-2B6-Ab1 3 mg/kg). A treatment withACI-36-2B6-Ab1 did not significantly affect total TAU and pTAU in the insarcosyl insoluble cortex homogenates. A slight decrease (withoutsignificance) of mean total TAU and p231TAU was observed uponACI-36-2B6-Ab1 treatment. TAU phosphorylation evaluated as the ratios ofpTAU to total TAU was not affected.

8.2.2.3 TAU in Soluble Fraction of Hippocampus Homogenates

Total TAU, p231TAU, p396TAU, and the ratios of pTAU to total TAU wereevaluated in the soluble fraction of hippocampus homogenates of n=16animals from group A (Tg vehicle group; PBS), B (Tg, ACI-36-2B6-Ab1, 1mg/kg), and C (Tg, ACI-36-2B6-Ab1, 3 mg/kg). treatment withACI-36-2B6-Ab1 did not significantly affect total TAU and pTAU in the insoluble hippocampus homogenates. A slight decrease (withoutsignificance) of TAU phosphorylation evaluated as the ratios of pTAU tototal TAU was observed upon ACI-36-2B6-Ab1 treatment.

8.2.2.4 TAU in Sarcosyl Insoluble Fraction of Hippocampus Homogenates

Total TAU, p231TAU, p396TAU, and the ratios of pTAU to total TAU wereevaluated in the sarcosyl insoluble fractions of hippocampus homogenatesof n=16 animals from group A (Tg vehicle group; PBS), B (Tg,ACI-36-2B6-Ab1, 1 mg/kg), and C (Tg, ACI-36-2B6-Ab1, 3 mg/kg). Atreatment with ACI-36-2B6-Ab1 did not significantly affect total TAU andpTAU in the in sarcosyl insoluble hippocampus homogenates. Treatmentwith 3 mg/kg increased the mean total TAU, p231TAU as well as p396TAU,although without reaching significance, whereas a slight reduction ofthe mean total TAU, p231TAU as well as p396TAU upon 1 mg/kg treatmentwas observed. TAU phosphorylation evaluated as the ratios of pTAU tototal TAU was slightly increased upon 3 mg/kg ACI-36-2B6-Ab1 treatment.

8.2.2.5. Western Blots for Soluble Cortex

Treatment with ACI-36-2B6-Ab1 dose-dependently reduced the presence ofboth the pS396/pS404 (FIG. 5D) and pT181 (FIGS. 5E and 5F) pTau epitopesin the soluble fraction of brain cortex, with a significant effect atthe 3 mg/kg dose.

8.2.2.6 TAU in biGT Tau Bigenic Mice

biGT mice treated with ACI-36-2B6-Ab1 for 3 months had significantlyreduced total Tau in brain cortex soluble fraction (FIGS. 6A and 6B). Asignificant reduction was observed for pTau epitopes pT231/AT180 (FIGS.6C and 6D), pS202/AT8 (FIG. 6E), and pS396 (FIGS. 6F and 6G). Asignificant reduction was also observed in both total homogenate (TH)for pTau epitope pS400 (FIGS. 6H and 6I) and pS404 (FIGS. 6L and 6M).Furthermore, a significant reduction was also observed in solublefraction for pTau epitopes pS400 (FIGS. 6J and 6K) and a trend forreduction for pTau epitope pS404 (FIGS. 6N and 6O).

8.2.3 Histology 8.2.3.1 Morphometry—Determination of Region Areas

Measured region areas of the hippocampus and the amygdala not differsignificantly throughout all investigated brains which excludes negativeeffects on tissue during dissection and IHC or staining (e.g.unequivocal shrinkage, different sectioning) and to a certain degreetreatment induced atrophy. Individual sections may deviate from theindividual and group mean because of e.g. folding of tissue or loss ofparts of the section during execution of the labeling protocol.Therefore, the total immunoreactive area [in μm2] of any labeling wasnormalized to the section's individual region area [in mm2] bycalculating the percentage of the labeled area within the region area[labeled area/(region area*10.000)].

8.2.3.2 Results of AT180 IH

The AT180 antibody detects the endogenous and human pTAU (doublyphosphorylated at Thr231 and Ser235). The amount of intrasomal pTAU innTg controls was significantly lower compared to Tg groups (p<0.01 aswell as p<0.001). In the amygdala, the higher dose of ACI-36-2B6-Ab1 (3mg/kg—group C) significantly decreased the somal pTAU compared tovehicle treated animals (FIG. 7, left). The lower dose (1 mg/kg—group C)showed the same tendency but did not reach significance. The same effectwas detectable in the hippocampus where ACI-36-2B6-Ab1 reduced pTAUdose-dependently, significant for the higher dose and tendentiously forthe lower (FIG. 7, right). This decrease was also qualitatively visibleas decrease of the area of staining and the staining intensity inindividual neuronal somata. Results of the sum staining intensitiesnormalized to A01 size of the measured AT180 IR in the neuronal somatawere comparable with measured AT180 IR area percentage, including asignificant dose-dependence of the greater effect of the higher dose inthe amygdala. In the hippocampus the post hoc comparisons did not reachsignificance level.

8.2.4 Summary

Brain Tau pathology as measured by MSD did not show a significantchange, however immunostaining of brain sections demonstrated adose-dependent with up to 60% reduction in AT180 (pT231/pS235)immunostaining in neuronal somata.

The study shows that passive immunization using thirteen administrationsof a phosphosite-specific anti-pTau antibody ACI-36-2B6-Ab1 can improvespatial learning and significantly reduces brain pTau pathology.

8.3 Results ACI-36-3A8-Ab1 Antibody

Thirteen i.p. injections of ACI-36-3A8-Ab1 administered weekly at 1 or 3mg/kg over a twelve week study period did not show any gross adverseeffects.

8.3.1 Behavioral Results—Morris Water Maze

The results from the MWM test demonstrated a significant effect ofimproved spatial learning for mice treated with ACI-36-3A8-Ab1 at 3mg/kg (FIG. 10).

Vehicle treated Tg (group A) and nTg (group F) control animals showedexpected learning curves in terms of escape latency and length of theswimming path to reach the platform over the four testing days. Tgcontrol (A) animals showed a significant impairment of learningabilities reflected in flatter learning curves of escape latencies andswimming paths compared to nTg control animals (group F). Escapelatencies and swimming paths were significantly (Two Way ANOVA)different on training days 3 (p<0.01, latency; p<0.001, length;Bonferroni's post test) and 4 (p<0.01; Bonferroni's post test).Treatment with ACI-36-3A8-Ab1, low or high dose (D and E) did notsignificantly improve spatial learning abilities compared to Tg controlanimals (A) when absolute values are analyzed. When adjusting theperformance of each group to 100% on training day 1 and all further daysas percentage of day 1, an improvement of learning and memory abilitiescan be upon ACI-36-3A8-Ab1 treatment (low and high dosage). Animalstreated with the low dose of ACI-36-3A8-Ab1 (group D) performed onlyslightly better in the MWM compared to Tg controls (group A). The effectof a weekly treatment with 3 mg/kg ACI-36-3A8-Ab1 (group E) was muchmore pronounced and almost restored the performance of nTg animals.Compared to the Tg controls (A) the effect of 3 mg/kg ACI-36-3A8-Ab1 wasstatistically significant for swimming path length on day 3 and day 4(p<0.05). No differences between treatment groups were detected whencalculating the swimming speed on all four training days.

In the probe trial (PT), the abidance in the target quadrant (south westquadrant) as well as target zone crossings were recorded. nTg controls(group F) spent more time in the target quadrant and crossed the targetzone more often relative to Tg controls (group A) but withoutstatistical significance. Treatment with neither the low nor the highdose led to a statistically significant improvement in comparison to Tgcontrol mice as evaluated in the PT. However, ACI-36-3A8-Ab1 treatedanimals had—although statistically insignificantly—more target zonecrossings compared to Tg control, that is in accordance with the outcomeof the swim length over the 4 training days.

8.3.2 Molecular Biology 8.3.2.1 TAU in Soluble Fraction of CortexHomogenates

Total TAU, p231TAU, p396TAU, and the ratios of pTAU to total TAU wereevaluated in the soluble fraction of cortex homogenates of n=16 animalsfrom group A (Tg vehicle group; PBS) and D (Tg, ACI-36-3A8-Ab1, 1 mg/kg)and of n=15 animals from group E (Tg, ACI-36-3A8-Ab1, 3 mg/kg). Atreatment with ACI-36-3A8-Ab1 did not significantly affect total TAU andpTAU in the in soluble cortex homogenates. However, a slight increase(without significance) of mean total TAU, p231TAU, and p396TAU wasobserved upon ACI-36-3A8-Ab1 treatment. TAU phosphorylation at 231evaluated as the ratio of p231TAU to total TAU was slightly decreasedafter treatment with 3 mg/kg ACI-36-3A8-AB1.

8.3.2.2 TAU in Sarcosyl Insoluble Fraction of Cortex Homogenates

Total TAU, p231TAU, p396TAU, and the ratios of pTAU to total TAU wereevaluated in the sarcosyl insoluble fractions of cortex homogenates ofn=16 animals from group A (Tg vehicle group; PBS) and D (Tg,ACI-36-3A8-Ab1, 1 mg/kg) and of n=15 animals from group E (Tg,ACI-36-3A8-Ab1, 3 mg/kg). A treatment with ACI-36-3A8-AB1 did notsignificantly affect total TAU and pTAU in the in sarcosyl insolublecortex homogenates. A slight decrease (without significance) of meantotal TAU, p231TAU, and p396TAU was observed upon 1 mg/ACI-36-3A8-Ab1treatment. Further the p231TAU to total TAU ratios of 1 mg/kgACI-36-3A8-Ab1 treated animals showed slightly lower variabilitycompared to vehicle treated animals (but with lacking significance inF-Test: p=0.184) without changing the mean p231TAU to total TAU ratiosof the two groups. For 1 mg/kg and 3 mg/kg ACI-36-3A8-Ab1 treated groupsa slight increase of p396TAU phosphorylation was observed.

8.3.2.3 TAU in Soluble Fraction of Hippocampus Homogenates

Total TAU, p231TAU, p396TAU, and the ratios of pTAU to total TAU wereevaluated in the soluble fraction of hippocampus homogenates of n=16animals from group A (Tg vehicle group; PBS) and D (Tg, ACI-36-3A8-Ab1,1 mg/kg) and of n=15 animals from group E (Tg, ACI-36-3A8-Ab1, 3 mg/kg).TAU and pTAU levels in the soluble hippocampus fractions of IRN6301(group D) were outliers and were excluded. A treatment withACI-36-3A8-Ab1 did not significantly affect total TAU and pTAU in the insoluble hippocampus homogenates. A slight decrease (withoutsignificance) of TAU phosphorylation at 231 evaluated as the ratios ofp231TAU to total TAU was observed upon ACI-36-3A8-Ab1 treatment.

8.3.2.4 TAU in Sarcosyl Insoluble Fraction of Hippocampus Homogenates

Total TAU, p231TAU, p396TAU, and the ratios of pTAU to total TAU wereevaluated in the sarcosyl insoluble fractions of hippocampus homogenatesof n=16 animals from group A (Tg vehicle group; PBS), B (TgACI-36-3A8-Ab1, 1 mg/kg), and C (Tg, ACI-36-3A8-Ab1, 3 mg/kg). Atreatment with ACI-36-3A8-Ab1 did not significantly affect total TAU andpTAU in the in sarcosyl insoluble hippocampus homogenates. A slightincrease (without significance) of mean total TAU, p231TAU as well asp396TAU was observed upon ACI-36-3A8-Ab1 treatment. TAU phosphorylationevaluated as the ratio of p231TAU to total TAU was not affected andtreatment with 1 mg/kg ACI-36-3A8-Ab1 slightly reduced the ratio ofp396TAU to total TAU.

8.3.2.5. Western Blots for Soluble Cortex

A significant reduction of the pS396/pS404 pTau epitope in the solublebrain cortex in mice treated with 1 or 3 mg/kg of ACI-36-3A8-Ab1 (FIG.5D). The presence of the human/transgenic pT181 pTau epitope was reducedin the soluble cortex fraction, with a significant effect in micetreated with 1 mg/kg and trend in mice treated with 3 mg/kg (FIG. 5E). Atrend for a reduction was observed in the amount of endogenous pT181pTau (FIG. 5F).

8.3.2.6 TAU in biGT Tau Bigenic Mice

biGT mice treated with ACI-36-3A8-Ab1 for 3 months had significantlyreduced total Tau in brain cortex soluble fraction (FIGS. 6A and 6B). Asignificant reduction was observed for pTau epitopes pT231/AT180 (FIGS.6C and 6D), pS202/AT8 (FIG. 6E), and pS396 (FIGS. 6F and 6G). Asignificant reduction was also observed in both total homogenate (TH)for pTau epitope pS400 (FIGS. 6H and 6I) and pS404 (FIGS. 6L and 6M).Furthermore, a significant reduction was also observed in solublefraction for pTau epitopes pS400 (FIGS. 6J and 6K) and a trend forreduction for pTau epitope pS404 (FIGS. 6N and 6O).

8.3.3 Histology 8.3.3.1 Morphometry—Determination of Region Areas

See Example 8.2.3.1

8.3.3.2 Results of AT180 IH

The AT180 antibody detects the endogenous and human pTAU (doublyphosphorylated at Thr231 and Ser235). The amount of intrasomal pTAU innTg controls was significantly lower compared to Tg groups (p<0.001). Inthe amygdala, both doses of ACI-36-3A8-Ab1 [1 mg/kg (group D) and 3mg/kg (group E)] significantly decreased the somal pTAU compared tovehicle treated animals (FIG. 8, left). A similar effect was detectablein the hippocampus where the lower dosage ACI-36-3A8-Ab1 reduced pTAU,however in this case the higher dose was less effective and just led toa tendentious decrease (FIG. 8, right). This decrease was alsoqualitatively visible as decrease of the area of staining and thestaining intensity in individual neuronal somata. Results of thenormalized sum of intensities of the measured AT180 IR in the neuronalsomata were comparable in the amygdala with measured AT180 IR areapercentage but reached significance for the higher dose only. In thehippocampus the result was totally comparable to IR area percentage.

8.3.4 Summary

Brain Tau pathology as measured by MSD did not show a significantchange, however immunostaining of brain sections demonstrated adose-dependent with up to 40% reduction in AT180 (pT231/pS235)immunostaining in neuronal somata.

The study shows that passive immunization using thirteen administrationsof a phosphosite-specific anti-pTau antibody ACI-36-3A8-Ab1 improvesspatial learning and significantly reduces brain pTau pathology.

Deposits

The following hybridoma cell lines were deposited in the name of ACImmune SA, PSE-EPFL Building B, 1015 Lausanne, Switzerland andKatholieke Universiteit Leuven, Minderbroedersstraat 8a—Box 5105, B-3000Leuven with the “Deutsche Sammlung von Mikroorganismen and ZellkulturenGmbH (DSMZ) in Braunschweig, Inhoffenstrasse 7 B, 38124 Braunschweig,under the provisions of the Budapest Treaty:

Hybridoma name Deposit number Date of deposit 6C10F9C12A11 DSM ACC3079Aug. 25, 2010 6C10E5E9C12 DSM ACC3081 Aug. 25, 2010 6H1A11C11 DSMACC3080 Aug. 25, 2010 6H1G6E6 DSM ACC3088 Aug. 25, 2010 2B6A10C11 DSMACC3084 Aug. 25, 2010 2B6G7A12 DSM ACC3087 Aug. 25, 2010 3A8A12G7 DSMACC3086 Aug. 25, 2010 3A8E12H8 DSM ACC3085 Aug. 25, 2010 7C2(1)F10C10D3DSM ACC3082 Aug. 25, 2010 7C2(2)B9F11D5 DSM ACC3083 Aug. 25, 2010A4-4A6-48 DSM ACC3136 Aug. 30, 2011 A6-2G5-30 DSM ACC3137 Aug. 30, 2011A6-2G5-41 DSM ACC3138 Aug. 30, 2011 A4-2A1-18 DSM ACC3139 Aug. 30, 2011A4-2A1-40 DSM ACC3140 Aug. 30, 2011 A6-1D2-12 DSM ACC3141 Sep. 6th, 2011

TABLE 1 Tau sequence, vaccine and antibody description Sequence*,Description Vaccine length (n), sequence ID number Hybridomas AntibodiesT1: Tau 5-20 [pY18] ACI-33 RQEFEVMEDHAGTY(p)GL (n = 16)  6C10F9C12A11ACI-33-6C10-Ab1 (SEQ ID NO: 59) 6C10E5E9C12 ACI-33-6C10-Ab2T8: Tau 206-221  ACI-41 PGSRSRT(p)PS(p)LPTPPTR (n = 16)  7C2(1)F10C10D3 ACI-41-7C2-Ab1 [pT212, pS214] (SEQ ID NO: 60) T9: Tau 196-211 GYSSPGS(p)PGT(p)PGSRSR (n = 16)  7C2(2)B9F11D5 ACI-41-7C2-Ab1[pS202, pT205] (SEQ ID NO: 61) T4: Tau 401-418  ACI-36GDTS(p)PRHLS(p)NVSSIGSID (n = 18)  6H1A11C11 ACI-36-6H1-Ab1[pS404, pS409] (SEQ ID NO: 63) 6H1G6E6 ACI-36-6H1-Ab2 2B6A10C11ACI-36-2B6-Ab1 2B6G7A12 ACI-36-2B6-Ab2 3A8A12G7 ACI-36-3A8-Ab1 3A8E12H8ACI-36-3A8-Ab2 T3: Tau 393-408  ACI-35 VYKS(p)PVVSGDTS(p)PRHL (n = 16) A4-4A6-48 ACI-35-4A6-Ab2 [pS396, pS404] (SEQ ID NO: 62) A6-2G5-30ACI-35-2G5-Ab2 A6-2G5-41 ACI-35-2G5-Ab3 A4-2A1-18 ACI-35-2A1-Ab1A4-2A1-40 ACI-35-2A1-Ab2 A6-1D2-12 ACI-35-1D2-Ab1 T5: Control sequence: ACI-37 RENAKAKTDHGAEIVYKS(p)PVVSGDTS(p)PRHL  Tau 379-408  (n =30) (SEQ ID NO: 58) [pS396, pS404] T8: Tau 206-221  ACI-39PGSRSRT(p)PS(p)LPTPPTR (n = 16)  [pT212, pS214] (SEQ ID NO: 60)T9: Tau 196-211  ACI-40 GYSSPGS(p)PGT(p)PGSRSR (n = 16)  [pS202, pT205](SEQ ID NO: 61) T2: Tau 200-216  ACI-34 PGS(p)PGT(p)PGSRSRT(p)PS(p)LP [pS202 + pT205 &  (n = 17) (SEQ ID NO: 64) pT212 + pS214]T10: Tau 407-418 ACI-42 HLS(p)NVSSTGSID (n = 12)  [pS409](SEQ ID NO: 65) T11: Tau 399-408 ACI-43 VSGDTS(p)PRHL (n = 10)  [pS404](SEQ ID NO: 66) *Based on the longest isoform of human Tau (Tau441). pindicates phosphorylated residue.

TABLE 2 Results of ACI-33 hybridoma screen T25 Flasks screen 24 wellplate screen Positive Positive in Positive in in IgG Positive Positivein ELISA TAUPIR screen in ELISA TAUPIR 1A7 1A7 1A11 1C11 1C11 2C9 2C93C3 3C3 3C3 3C5 3C5 3E8 3E8 3G10 3G10 3G10 3G10 6C10 6C10 6C10 6C10 6C106F3 6F3 6F8 6F8

TABLE 3 Results of ACI-36 hybridoma screen T25 Flasks screen 24 wellplate screen Positive Positive in Positive in in IgG Positive Positivein ELISA TAUPIR screen in ELISA TAUPIR 2B6 2B6 2B6 2B6 2B6 2F9 2F9 2F92F9 2F9 2G1 2G1 2G1 2G1 3A8 3A8 3A8 3A8 3A8 3B9 3B9 3B9 3B9 3F11 3F113F11 3F11 4A3 4A3 4C1 4C1 4C1 4C1 4C12 4C12 4C12 4C12 4E12 4E12 4E124E12 5E10 5E10 5E10 5F5 5F5 5F5 7D6 7D6 7D6 7D6 7D6 6H1 6H1 6H1 6H1

TABLE 4 Ranking for positive clones in ELISA and TAUPIR of ACI-36ranking for ELISA ranking for TAUPIR 3A8 6H1 2B6 4C1 4C1 3A8 6H1 4C124C12 2B6 2G1 2F9 2F9 3B9 7D6 2G1 3B9 7D6 4E12 4E12

TABLE 5 Results of ACI-41 hybridoma screen T25 Flasks screen 24 wellplate screen Positive Positive Positive in Positive in in IgG Positivein ELISA TAUPIR screen in ELISA TAUPIR 3D11 3D11 3D11 4H6 4H6 4H6 5D105D10 5D10 5D10 5D10 5E6 5E6 5F10 5F10 6B7 6B7 6B7 7C2 7C2 7C2 7C2 7C28G8 8G8 8H8 8H8 8H8

TABLE 6 Screening of hybridomas for binding to target ELISA Full- Full-Tau p- Tau length length Western Hybridomas Antibodies peptide peptidepTau Tau TAUPIR Blot 6C10F9C12A11 ACI-33- + − +/− − + − 6C10-Ab16C10E5E9C12 ACI-33- + − +/− − + − 6C10-Ab2 6H1A11C11 ACI-36- + − + − + +6H1-Ab1 6H1G6E6 ACI-36- + − + − + + 6H1-Ab2 2B6A10C11 ACI-36- + − +− + + 2B6-Ab1 2B6G7A12 ACI-36- + − + − + + 2B6-Ab2 3A8A12G7 ACI-36- +− + − + + 3A8-Ab1 3A8E12H8 ACI-36- + − + −/+ + + 3A8-Ab2 7C2(1)F10C10D3ACI-41- + − + − + − 7C2-Ab1 7C2(2)B9F11D5 ACI-41- + − + − + − 7C2-Ab2A4-2A1-18 ACI-35- + − + − 2A1-Ab1 A4-2A1-40 ACI-35- + − + − 2A1-Ab2A4-4A6-18 ACI-35- + − − + 4A6-Ab1 A4-4A6-48 ACI-35- 4A6-Ab2 A6-1D2-12ACI-35- + − + − 1D2-Ab1 A6-2G5-08 ACI-35- + − − − 2G5-Ab1 A6-2G5-30ACI-35- + − + − 2G5-Ab2 A6-2G5-41 ACI-35- + − + − 2G5-Ab3

TABLE 7 Binding affinity of anti-tau antibodies Association rateDissociation constant rate Dissociation (k_(d)) constant constantHybridomas Antibodies (1/Ms) (k_(a)) (1/s) (K_(D)) (nM) 6C10F9C12AACI-33-6C10-Ab1 9.46 × 10⁵ 3.27 × 10⁻³ 3.46 11 6H1A11C11 ACI-36-6H1-Ab13.53 × 10⁴ 6.80 × 10⁻⁵ 1.93 6H1G6E6 ACI-36-6H1-Ab2 9.99 × 10⁴ 9.58 ×10⁻⁵ 0.96 2B6A10C11 ACI-36-2B6-Ab1 6.90 × 10⁵ 1.63 × 10⁻⁴ 0.24 2B6G7A12ACI-36-2B6-Ab2 9.11 × 10⁵ 1.11 × 10⁻⁴ 0.12 3A8A12G7 ACI-36-3A8-Ab1 1.01× 10⁶ 1.09 × 10⁻⁴ 0.11 3A8E12H8 ACI-36-3A8-Ab2 8.43 × 10⁵ 1.43 × 10⁻⁴0.17 A4-4A6-18 ACI-35-4A6-Ab1 2.00 × 10⁵ 3.10 × 10⁻³ 16 A6-1D2-12ACI-35-1D2-Ab1 1.60 × 10³ 9.30 × 10⁻⁶ ≦6 A6-2G5-08 ACI-35-2G5-Ab1 4.80 ×10⁵ 5.30 × 10⁻³ 10

TABLE 8 Peptide libraries used for epitope mapping Peptide library forT1 Tau(441) amino acid number 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Amino acid Peptide no R Q E F E V M E D H A G T Y(P) G L Phosphopeptides T1.18 A G T Y(P) G L T1.17 H A G T Y(P) G L T1.16 D H A G TY(P) G L T1.15 E D H A G T Y(P) G L T1.14 M E D H A G T Y(P) G L T1.13 VM E D H A G T Y(P) G L T1.12 E V M E D H A G T Y(P) G L T1.11 F E V M ED H A G T Y(P) G L T1.10 E F E V M E D H A G T Y(P) G L T1.9 Q E F E V ME D H A G T Y(P) G L T1.7 R Q E F E V M E D H A G T Y(P) G L Amino acidPeptide no R Q E F E V M E D H A G T Y G L Non-phospho peptides T1.28 AG T Y G L T1.27 H A G T Y G L T1.26 D H A G T Y G L T1.25 E D H A G T YG L T1.24 M E D H A G T Y G L T1.23 V M E D H A G T Y G L T1.22 E V M ED H A G T Y G L T1.21 F E V M E D H A G T Y G L T1.20 E F E V M E D H AG T Y G L T1.19 Q E F E V M E D H A G T Y G L T1.8 R Q E F E V M E D H AG T Y G L Peptide library for T4 Tau(441) amino acid number 401 402 403404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 PeptideAmino acid no. G D T S(p) P R H L S(p) N V S S T G S I D Phosphopeptides T3.17 G D T S(p) P R H L T4.11 D T S(p) P R H L S(p) T4.12 TS(p) P R H L S(p) N T4.13 S(p) P R H L S(p) N V T4.14 P R H L S(p) N V ST4.15 R H L S(p) N V S S T4.16 H L S(p) N V S S T T4.17 L S(p) N V S S TG T4.18 S(p) N V S S T G S T4.19 N V S S T G S I T4.20 V S S T G S I DPeptide Amino acid no. G D T S P R H L S N V S S T G S I D Non-phosphopeptides T3.26 G D T S P R H L T4.21 D T S P R H L S T4.22 T S P R H L SN T4.23 S P R H L S N V T4.24 P R H L S N V S T4.25 R H L S N V S ST4.26 H L S N V S S T T4.27 L S N V S S T G T4.28 S N V S S T G S T4.19N V S S T G S I T4.20 V S S T G S I D Peptide library for T8 Tau(441)amino acid number 206 207 208 209 210 211 212 213 214 215 216 217 218219 220 221 Amino acid Peptide no P G S R S R T(p) P S(p) L P T P P T RPhospho peptides T8.7 P G S R S R T(p) P T8.8 G S R S R T(p) P S(p) T8.9S R S R T(p) P S(p) L T8.10 R S R T(p) P S(p) L P T8.11 S R T(p) P S(p)L P T T8.12 R T(p) P S(p) L P T P T8.13 T(p) P S(p) L P T P P T8.14 PS(p) L P T P P T T8.15 S(p) L P T P P T R Amino acid Peptide no P G S RS R T P S L P T P P T R Non-phospho peptides T8.16 P G S R S R T P T8.17G S R S R T P S T8.18 S R S R T P S L T8.19 R S R T P S L P T8.20 S R TP S L P T T8.21 R T P S L P T P T8.22 T P S L P T P P T8.23 P S L P T PP T T8.24 S L P T P P T R Peptide library for T9 Tau(441) amino acidnumber 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211Amino acid Peptide no G Y S S P G S(p) P G T(p) P G S R S R Phosphopeptides T9.7 G Y S S P G S(p) P T9.8 Y S S P G S(p) P G T9.9 S S P GS(p) P G T(p) T9.10 S P G S(p) P G T(p) P T9.11 P G S(p) P G T(p) P GT9.12 G S(p) P G T(p) P G S T9.13 S(p) P G T(p) P G S R T9.14 P G T(p) PG S R S T9.15 G T(p) P G S R S R Amino acid Peptide no G Y S S P G S P GT P G S R S R Non-phospho peptides T9.16 G Y S S P G S P T9.17 Y S S P GS P G T9.18 S S P G S P G T T9.19 S P G S P G T P T9.20 P G S P G T P GT9.21 G S P G T P G S T9.22 S P G T P G S R T9.23 P G T P G S R S T9.24G T P G S R S R Peptide library for T3 Tau(441) amino acid number 393394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 PeptideAmino acid no. V Y K S(p) P V V S G D T S(p) P R H L Phospho peptidesT3.9 V Y K S(p) P V V S T3.10 Y K S(p) P V V S G T3.11 K S(p) P V V S GD T3.12 S(p) P V V S G D T T3.13 P V V S G D T S(p) T3.14 V V S G D TS(p) P T3.15 V S G D T S(p) P R T3.16 S G D T S(p) P R H T3.17 G D TS(p) P R H L Peptide Amino acid no. V Y K S P V V S G D T S P R H LNon-phospho peptides T3.18 V Y K S P V V S T3.19 Y K S P V V S G T3.20 KS P V V S G D T3.21 S P V V S G D T T3.22 P V V S G D T S T3.23 V V S GD T S P T3.24 V S G D T S P R T3.25 S G D T S P R H T3.26 G D T S P R HL

TABLE 9 Tau amino acids and phospho-residues required for antibodybinding. Vaccine Hybridoma Epitope* ACI-33 6C10F9C12A11 Tau aa 15-20,with requirement for pY18 ACI-33 6C10E5E9C12 Tau aa 15-20, withrequirement for pY18 ACI-36 6H1A11C11 Tau aa 405-412, with requirementfor pS409 ACI-36 6H1G6E6 Tau aa 405-412, with requirement for pS409ACI-36 2B6A10C11 Tau aa 405-411, with requirement for pS409 ACI-362B6G7A12 Tau aa 405-411, with requirement for pS409 ACI-36 3A8A12G7 Tauaa 405-411, with requirement for pS409 ACI-36 3A8E12H8 Tau aa 405-411,with requirement for pS409 ACI-41 7C2(1) Tau aa 208-218, withrequirement for pT212 F10C10D3 and pS214 ACI-35 A4-2A1-18 Tau aa393-401, with requirement for pS396 ACI-35 A4-2A1-40 Tau aa 393-401,with requirement for pS396 ACI-35 A4-4A6-18 Tau aa 396-401, withrequirement for pS396 ACI-35 A6-1D2-12 Tau aa 394-400, with requirementfor pS396 ACI-35 A6-2G5-08 Tau aa 402-406, with requirement for pS404ACI-35 A6-2G5-30 Tau aa 393-400, with requirement for pS396 ACI-35A6-2G5-41 Tau aa 393-400, with requirement for pS396 *Based on thelongest isoform of human Tau (Tau441)

TABLE 10Amino Acid Sequence of the heavy chain and light chain variable regions (VH and VK) and the CDRsVH Primer VK Primer Vaccine Hybridoma (mix) (mix) VH VK VH CDR1 VH CDR2VH CDR3 VK CDR1 VK CDR2 VK CDR3 ACI-36 3A8Al2G7* SEQ ID VK_ADEVQLQQSGPELVKPGA DIVMTQSPSSLAMSVG GYTFTD DINPNR YYAVG KSSQSVF FASTREQEHYTT NO: 46 (SEQ ID SVKISCKASGYTFTDYY QKVTMSCKSSQSVFNS YYMN GGTTYNY (SEQ NSGNQK S (SEQ PPT and NO: 48/ MNWVKQSHGKSLEWIG GNQKNSLAWYQQKPG(SEQ ID QKFKG ID NO: NSLA ID NO: (SEQ ID SEQ ID SEQ ID NO:DINPNRGGTTYNQKFK QSPKLLVYFASTRESGV NO: 12) (SEQ ID 14) (SEQ ID 22)NO: 23) NO: 47 49/ and GKATLTVDKSSSTAYM PDRFIGSGSGTDFSLTI NO: 13)NO: 21) SEQ ID NO: ELRSLTSEDSAVYYCAS SSVQAEDLADYFCQEH 51)YYAVGYWGQGTTLTVS YTTPPTFGTGTKLELK S (SEQ ID NO: 1) (SEQ ID NO: 6) ACI-363A8A12G7* SEQ ID VK_G EVQLQQSGPELVKPGA DVVMTQTPLSLPVSLG GYTFTD DINPNRYYAVG RSSQRLV KVSNRF SQTAHF NO: 46 (SEQ ID SVKISCKASGYTFTDYYDQASISCRSSQRLVHS YYMN GGTTYN Y (SEQ HSHGKT S (SEQ PYT and NO: 50 andMNWVKQSHGKSLEWIG HGKTYLHWYLQKPGQS (SEQ ID QKFKG ID NO: YLH (SEQ ID NO:(SEQ ID SEQ ID SEQ ID NO: DINPNRGGTTYNQKFK PKLLIYKVSNRFSGVPD NO: 12)(SEQ ID 14) ID NO: 24) 25) NO: 26) NO: 47 51) GKATLTVDKSSSTAYMRFSGSGSGTDFTLKISR NO: 13) ELRSLTSEDSAVYYCAS VEAEDLGVYFCSQTAHYYAVGYWGQGTTLTVS FPYTFGGGTKLEIK S (SEQ ID NO: 1 (SEQ ID NO: 7) ACI-363A8E12H8* SEQ ID VK_AD EVQLQQSGPELVKPGA DIVMTQSPSSLAMSVG GYTFTD DINPNRYYAVG KSSQSVF FASTRE QEHYTT NO: 46 (SEQ ID SVKISCKASGYTFTDYYQKVTMSCKSSQSVFNS YYMN GGTTYN Y (SEQ NSGNQK S (SEQ PPT and NO: 48/MNWVKQSHGKSLEWIG GNQKNSLAWYQQKPG (SEQ ID QKFKG ID NO: NSLA ID NO:(SEQ ID SEQ ID SEQ ID NO: DINPNRGGTTYNQKFK QSPKLLVYFASTRESGV NO: 12(SEQ ID 14) (SEQ ID 22) NO: 23) NO: 47 49 and SEQ GKATLTVDKSSSTAYMPDRFIGSGSGTDFSLTI NO: 13) NO: 21) ID NO: 51) ELRSLTSEDSAVYYCASSSVQAEDLADYFCQEH YYAVGYWGQGTTLTVS YTTPPTFGTGTKLELK S (SEQ ID NO: 1)(SEQ ID NO: 6) ACI-36 3A8E12H8* SEQ ID VK_G EVQLQQSGPELVKPGADVVMTQTPLSLPVSLG GYTFTD DINPNR YYAVG RSSQRLV KVSNRF SQTAHF NO: 46(SEQ ID SVKISCKASGYTFTDYY DQASISCRSSQRLVHS YYMN GGTTYN Y (SEQ HSHGKTS (SEQ PYT and NO: 50 and MNWVKQSHGKSLEWIG HGKTYLHWYLQKPGQS (SEQ IDQKFKG ID NO: YLH (SEQ ID NO: (SEQ ID SEQ ID SEQ ID NO: DINPNRGGTTYNQKFKPKLLIYKVSNRFSGVPD NO: 12 (SEQ ID 14) ID NO: 24) 25) NO: 26) NO: 47 51)GKATLTVDKSSSTAYM RFSGSGSGTDFTLKISR NO: 13) ELRSLTSEDSAVYYCASVEAEDLGVYFCSQTAH YYAVGYWGQGTTLTVS FPYTFGGGTKLEIK S (SEQ ID NO: 1)(SEQ ID NO: 7) ACI-36 2B6A10C11 SEQ ID SEQ ID NO: EVQLQQSGPELVKPGTDVVMTQTPLSLPVSLG GYTFTD DINPNR YYAVG RSSQSLV KVSNRF SQTAHF NO:50 and SEQ SVKISCKASGYTFTDYY DQASISCRSSQSLVHSH YYMN GGTTYN Y (SEQ HSHGKTS (SEQ PYT 46/ SEQ ID NO: 51 MNWVKQSHGKSLEWIG GKTYLHWYLQKPGQSP (SEQ IDQKFKG ID NO: YLH (SEQ ID NO: (SEQ ID ID NO: DINPNRGGTTYNQKFKKLLIYKVSNRFSGVPDR NO: 12) (SEQ ID 14) ID NO: 27) 25) NO: 26) 52 andGKATLTVDKSSSTAYM FSGSGSGTDFTLKISRV NO: 13) SEQ ID ELRSLTSEDSAVYYCASEAEDLGVYFCSQTAHF NO: 47 YYAVGYWGQGTTLTVS PYTFGGGTKLEIK  S (SEQ ID NO: 2)(SEQ ID NO: 8) ACI-36 2B6G7Al2 SEQ ID SEQ ID NO: EVQLQQSGPELVKPGTDVVMTQTPLSLPVSLG GYTFTD DINPNR YYAVG RSSQSLV KVSNRF SQTAHF NO:50 and SEQ SVKISCKASGYTFTDYY DQASISCRSSQSLVHSH YYMN GGTTYN Y (SEQ HSHGKTS (SEQ PYT 46/ SEQ ID NO: 51 MNWVKQSHGKSLEWIG GKTYLHWYLQKPGQSP (SEQ IDQKFKG ID NO: YLH (SEQ ID NO: (SEQ ID ID NO: DINPNRGGTTYNQKFKKLLIYKVSNRFSGVPDR NO: 12) (SEQ ID 14) ID NO: 27) 25) NO: 26) 52 andGKATLTVDKSSSTAYM FSGSGSGTDFTLKISRV NO: 13) SEQ ID ELRSLTSEDSAVYYCASEAEDLGVYFCSQTAHF NO: 47 YYAVGYWGQGTTLTVS PYTFGGGTKLEIK  S (SEQ ID NO: 2)(SEQ ID NO: 8) ACI-36 6H1A11C11 SEQ ID SEQ ID NO: EVQLQQSGPELVKPGADVVMTQTPLSLPVSLG GYTFTD DINPNR YYAVG RSSQSLL KVSNRF SQTAHF NO: 4650 and SEQ SVKISCKASGYTFTDYY DQASISCRSSQSLLHSH YYMN GGTTYN Y (SEQ HSHGNTS (SEQ PYT and ID NO: 51 MNWVKQSHGKSLEWIG GNTYLHWYLQKPGQSP (SEQ ID QKFKGID NO: YLH (SEQ ID NO: (SEQ ID SEQ ID DINPNRGGTTYNQKFK KLLIYKVSNRFSGVPDRNO: 12) (SEQ ID 14) ID NO: 28) 25) NO: 26)) NO: 47 GKATLTVDTSSSTAYMEFSGSGSGTDFTLKISRV NO: 13) LRSLTSEDSAVYYCASY EAEDLGVYFCSQTAHFYAVGYWGQGTTLTVSS PYTFGGGTKLEIK  (SEQ ID NO: 3) (SEQ ID NO: 9) ACI-366H1G6E6 SEQ ID SEQ ID NO: EVQLQQSGPELVKPGA DVVMTQTPLSLPVSLG GYTFTDDINPNR YYAVG RSSQSLL KVSNRF SQTAHF NO: 46 50 and SEQ SVKISCKASGYTFTDYYDQASISCRSSQSLLHSH YYMN GGTTYN Y (SEQ HSHGNT (SEQ ID PYT and ID NO: 51MNWVKQSHGKSLEWIG GNTYLHWYLQKPGQSP (SEQ ID QKFKG ID NO: YLH (SEQ NO: 25)(SEQ ID SEQ ID DINPNRGGTTYNQKFK KLLIYKVSNRFSGVPDR NO: 12) (SEQ ID 14)ID NO: 28) NO: 26) NO: 47 GKATLTVDTSSSTAYME FSGSGSGTDFTLKISRV NO: 13)LRSLTSEDSAVYYCASY EAEDLGVYFCSQTAHF YAVGYWGQGTTLTVSS PYTFGGGTKLEIK (SEQ ID NO: 3) (SEQ ID NO: 9) ACI-33 6C10E5E9C SEQ ID SEQ ID NO:EVQLVESGGGLVKPGG DIVMTQSHKFMSTSVG GFTFSD YISSGSS RGQLR KASQDV SASYRYQQHYTT 12 NO: 48/ SEQ ID SLKLSCAPSGFTFSDYG DRVSITCKASQDVSTAV YGMHTIYYGDT LRLFAY STAVA T (SEQ PLT (SEC 53/ SEQ NO: 49 and MHWVRQAPEKGLEWVAWYQQKPGQSPKLLIY (SEQ ID VKG (SEQ ID (SEQ ID ID NO: ID NO: ID SEQ ID NO:AYISSGSSTIYYGDTVK SASYRYTGVPDRFTGS NO: 15) (SEQ ID NO: 17) NO: 29) 30)31) NO: 54 51 GRFTISRDNAKNTLFLQ GSGTDFTFTISSVQAED NO: 16) andMTSLRSEDTAMYYCAR LAVYYCQQHYTTPLTFG SEQ ID RGQLRLRLFAYWGQGTAGTKLELK (SEQ ID NO: 47 LVTVSA  NO: 10) (SEQ ID NO: 4) ACI-33 6C10F9C12SEQ ID SEQ ID NO: EVQLVESGGGLVKPGG DIVMTQSHKFMSTSVG GFTFSD YISSGSS RGQLRKASQDV SASYRY QQHYTT A11 NO: 54 and SEQ SLKLSCAPSGFTFSDYGDRVSITCKASQDVSTAV YGMH TIYYGDT LRLFAY STAVA T (SEQ PLT (SEC 53/ SEQID NO: 51 MHWVRQAPEKGLEWV AWYQQKPGQSPKLLIY (SEQ ID VKG (SEQ ID (SEQ IDID NO: ID NO: ID AYISSGSSTIYYGDTVK SASYRYTGVPDRFTGS NO: 15) (SEQ IDNO: 17) NO: 29) 30) 31) NO: 54 GRFTISRDNAKNTLFLQ GSGTDFTFTISSVQAEDNO: 16) and MTSLRSEDTAMYYCAR LAVYYCQQHYTTPLTFG SEQ ID RGQLRLRLFAYWGQGTAGTKLELK  NO: 47 LVTVSA  (SEQ ID NO: 10) (SEQ ID NO: 4) ACI-417C2(1)F10C SEQ ID SEQ ID NO: EVKLMESGGGLVHPGA DIVMSQSPSSLAVSVGE GFTFTDLIRNKAN ALGRY KSSQSLL WASTRE QQYYSY 10D3 NO: 53/ 49/SEQ IDSLRLYCAASGFTFTDYY KVTMSCKSSQSLLYSS YYMS GYTTEY FDV YSSNQK S (SEQ PFTSEQ ID NO: 56/ SEQ MSWVRQPPGKAPEWL NQKNYLAWYQQKPGQ (SEQ ID TASVKG(SEQ ID NYLA ID NO: (SEQ ID NO: 55 ID NO: 57 ALIRNKANGYTTEYTASSPKLLIYWASTRESGVP NO: 18) (SEQ ID NO: 20) (SEQ ID 33) NO: 34) andand SEQ ID VKGRFTISRDNSQNILY DRFTGSGSGTDFTLTIS NO: 19) NO: 32) SEQ IDNO: 51 LQMNTLRAEDSATYYC SVKAEDLAVYYCQQYY NO: 47 VKALGRYFDVWGTGTTSYPFTFGSGTKLEIK VTVSS (SEQ ID NO: 11) (SEQ ID NO: 5) ACI-41 7C2(2)B9F1SEQ ID SEQ ID NO: EVKLMESGGGLVHPGA DIVMSQSPSSLAVSVGE GFTFTD LIRNKANALGRY KSSQSLL WASTRE QQYYSY 1D5 NO: 53/ 57 and SEQ SLRLYCAASGFTFTDYYKVTMSCKSSQSLLYSS YYMS GYTTEY FDV YSSNQK S (SEQ PFT SEQ ID ID NO: 51MSWVRQPPGKAPEWL NQKNYLAWYQQKPGQ (SEQ ID TASVKG (SEQ ID NYLA ID NO:(SEQ ID NO: 55 ALIRNKANGYTTEYTAS SPKLLIYWASTRESGVP NO: 18) (SEQ IDNO: 20) (SEQ ID 33) NO: 34) and VKGRFTISRDNSQNILY DRFTGSGSGTDFTLTISNO: 19) NO: 32) SEQ ID LQMNTLRAEDSATYYC SVKAEDLAVYYCQQYY NO: 47VKALGRYFDVWGTGTT SYPFTFGSGTKLEIK VTVSS  (SEQ ID NO: 11) (SEQ ID NO: 5)ACI-35 A4-4A6-18 QVQLQQPGAELLKPGA DVLMTQTPLSLPVSLGD GYTFTS RIDPNS DDYAWRSSQSIV KLSNRF FQGSHV SVKLSCKASGYTFTSY QASISCRSSQSIVHSNG YWMH DRTKYN FAYHSNGNT S PPT WMHWVKQRPGRGLE NTYLEWYLQKPGQSPK (SEQ ID  EKFKR (SEQ ID YLE(SEQ ID (SEQ ID WIGRIDPNSDRTKYNEK LLIYKLSNRFSGVPDRF NO: 70 (SEQ IDNO: 72) (SEQ ID NO: 74) NO: 75) FKRKATLTVDKSSSTAY SGSGSGTDFTLKISRVENO: 71) NO: 73) MQLSSLTSEDSAVYYC AEDLGVYYCFQGSHVP ARDDYAWFAYWGQGTPTFGGGTKLEIK (SEQ LVTVSA  ID NO: 69) (SEQ ID NO: 68) ACI-35 A6-1D2-12QVTLKESGPGILQSSQT NILMTQSPSSLAVSAGE GFSLST HIYWDD LLRPYA KSSQSVL WASTRELQYLSSL LSLTCSFSGFSLSTSGM KVTMSCKSSQSVLYSS SGMGVS DKRYNA LDY YSSNQK S TGVSWIRQPSGKGLEWL NQKNYLAWYQQKPGQ (SEQ ID SLKS (SEQ ID NYLA (SEQ ID(SEQ ID AHIYWDDDKRYNASLK SPKLLIYWASTRESGVP NO: 78) (SEQ ID NO: 80)(SEQ ID NO: 82) NO: 83) SRLTISKDTSRNQVFLKI DRFTGSGSGTDFTLTIS NO: 79)NO: 81) TCVDTADTATYYCARLL SVQAEDLAVYYCLQYLS RPYALDYWGQGTSVTVSLTFGAGTKLELK (SEQ SS (SEQ ID NO: 76) ID NO: 77) ACI-35 A4-2A1-18EVQLQQSGPELVKPGA DIVMTQAAPSVPVTPGE GYTFTD DINPNN EGRFA RSSKSLL RMSNLAMQHLKS SVKISCKASGYTFTDYY SVSISCRSSKSLLHSNG YYMN GGTSYN Y HSNGNT S PYTMNWVKQSHGKSLEWIG NTYLYWFLQRPGQSPQ (SEQ ID QKFKG (SEQ ID YLY (SEQ ID(SEQ ID DINPNNGGTSYNQKFK LLIHRMSNLASGVPDRF NO: 89) (SEQ ID  NO: 91)(SEQ ID NO: 94) NO: 95) GKATLTVDKSSSTAYM SGSGSGTAFTLRISRVE NO: 90)NO: 93) ELRSLTSEDSAVYYCVR AEDVGVYYCMQHLKSP EGRFAYWGHGTLVTVSYTFGGGTKLEIK (SEQ A (SEQ ID NO: 88) ID NO: 116) ACI-35 A4-2A1-40EVQLQQSGPELVKPGA DIX*MTQAAPSVPVTPG GYTFTD DINPNN EGRFA RSSKSLL RMSNLAMQHLKS SVKISCKASGYTFTDYY ESVSISCRSSKSLLHSN YYMN GGTSYN Y HSNGNT S PYTMNWVKQSHGKSLEWIG GNTYLYWFLQRPGQSP (SEQ ID QKFKG (SEQ ID YLY (SEQ ID(SEQ ID DINPNNGGTSYNQKFK QLLIYRMSNLASGVPDR NO: 89) (SEQ ID  NO: 91)(SEQ ID NO: 94) NO: 95) GKATLTVDKSSSTAYM FSGSGSGTAFTLRISRV NO: 90)NO: 93) ELRSLTSEDSAVYYCVR EAEDVGVYYCMQHLKS EGRFAYWGHGTLVTVSPYTFGGGTKLEIK (SEQ A (SEQ ID NO: 88) ID NO: 92) *X = M or V ACI-35A4-4A6-48 EVQLQQSGPELVKPGA DIVMTQAAPSVPVTPGE GYTFTD DINPNN EGRFA RSSKSLLRMSNLA MQHLKS SVKISCKASGYTFTDYY SVSISCRSSKSLLHSNG YYMN GGTSYN Y HSNGNT SPYT MNWVKQSHGKSLEWIG NTYLYWFLQRPGQSPQ (SEQ ID QKFKG (SEQ ID YLY (SEQ ID(SEQ ID DINPNNGGTSYNQKFK LLIYRMSNLASGVPDRF NO: 89) (SEQ ID  NO: 91) (SEQ ID NO: 94) NO: 95) GKATLTVDKSSSTAYM SGSGSGTAFTLRISRVE NO: 90)NO: 93) ELRSLTSEDSAVYYCVR AEDVGVYYCMQHLKSP EGRFAYWGHGTLVTVSYTFGGGTKLEIK (SEQ A (SEQ ID NO: 88) ID NO: 118) ACI-35 A6-2G5-08QVQLKQSGAELVRPGA DVLMTQTPLSLPVSLGD GYTFTD RIYPGR FWDVT RSSQSIV KVSNRFFQGSHV SVKLSCKASGYTFTDYY QASISCRSSQSIVHSNG YYIN GNIYYN Y HSNGNT S PYTINWVKQRPGQGLEWIA NTYLEWFLQKPGQSPK (SEQ ID EKFKG (SEQ ID  YLE (SEQ ID(SEQ ID RIYPGRGNIYYNEKFKG LLIYKVSNRFSGVPDRF NO: 98) (SEQ ID NO: (SEQ IDNO: 102) NO: 103) KATLTAEKSSSTAYMQL SGSGSGTDFTLKISRVE NO: 99)  100)NO: 101) SSLTSEDSAVYFCARF AEDLGVYYCFQGSHVP WDVTYWGQGTLVTVSA YTFGGGTKLEIK(SEQ ID NO: 96) (SEQ ID NO: 97) ACI-35 A6-2G5-30 EVQLQQSGPELVKPGADIVMTQSQKFMSTSVG GFTFTD DINPNN EGRFA KASQNV SASYRY QQYNSYSVKISCKASGFTFTDYY DRVSVTCKASQNVGTN YYMN GGTSYH Y GTNVA S PYTMNWVKQSHGKSLEWIG VAWYQQKPGQSPKALI (SEQ ID QKFKG (SEQ ID (SEQ ID  (SEQ ID(SEQ ID DINPNNGGTSYHQKFK YSASYRYSGVPDRFTG NO: 89) (SEQ ID  NO: 91)NO: 106) NO: 107) NO: 108) GKATLTVDKSSSTAYM SGSGTDFTLTISNVQSE NO: 115)ELRSLTSEDSAVYYCVR DLAEYFCQQYNSYPYT EGRFAYWGQGTLVTVS FGGGTKLEIKA (SEQ ID NO: 104) (SEQ ID NO: 105) ACI-35 A6-2G5-41 EVQLQQSGPELVKPGADIVMTQSQKFMSTSVG GFTFTD DINPNN EGRFA KASQNV SASYRY QQYNSYSVKISCKASGFTFTDYY DRVSVTCKASQNVGTN YYMN GGTSYH Y GTNVA S PYTMNWVKQSHGKSLEWIG VAWYQQKPGQSPKALI (SEQ ID  QKFKG (SEQ ID (SEQ ID(SEQ ID  (SEQ ID DINPNNGGTSYHQKFK YSASYRYSGVPDRFTG NO: 89) (SEQ IDNO: 91) NO: 106) NO: 107) NO: 108) GKATLTVDKSSSTAYM SGSGTDFTLTISNVQSENO: 115) ELRSLTSEDSAVYYCVR DLAEYFCQQYNSYPYT EGRFAYWGQGTLVTVS FGGGTKLEIKA (SEQ ID NO: 104) (SEQ ID NO: 105) *Two productive VK sequencessequences 6 and 7 in Table 10; sequences 40 and 41 in Table 11) wereisolated from cell lines 3A8Al2G7 and 3A8E12H8; the ″V_(K)_G″ sequenceswere prepared from clones made using ″G″ primer mix and ″V_(K)_AD″sequences from clones made using ″A″ and ″D″ primer mixes. Accordingly,two antibodies with different kappa sequences are produced by thesehybridomas.

TABLE 11 Amino Acid Sequence of the hevy chain and light chain variableregions (VH and VK) and CDRs VH VK Primer Primer Vaccine Hybridoma (mix)(mix) VH ACI-36 3A8A12G7* SEQ ID VK_ADGAGGTCCAGCTGCAACAATCTGGACCTGAACTGGTG NO: 46 (SEQAAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCT and ID NO:TCTGGATATACGTTCACTGACTACTACATGAACTGGG SEQ ID 48/TGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTG NO: 47 SEQ IDGAGATATTAATCCTAACCGTGGTGGAACTACTTACAA NO: CCAGAAGTTCAAGGGCAAGGCCACGTTGACTGTAGA 49/ CAAGTCCTCCAGCACAGCCTACATGGAACTCCGCAG andCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCA SEQ IDAGTTACTACGCCGTGGGCTACTGGGGCCAAGGCAC NO: 51)CACTCTCACAGTCTCCTCA (SEQ ID NO: 35) ACI-36 3A8Al2G7* SEQ ID VK_GGAGGTCCAGCTGCAACAATCTGGACCTGAACTGGTG NO: 46 (SEQAAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCT and ID NO:TCTGGATATACGTTCACTGACTACTACATGAACTGGG SEQ ID 50 andTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTG NO: 47 SEQ IDGAGATATTAATCCTAACCGTGGTGGAACTACTTACAA NO: 51) CCAGAAGTTCAAGGGCAAGGCCACGTTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAACTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGTTACTACGCCGTGGGCTACTGGGGCCAAGGCAC CACTCTCACAGTCTCCTCA (SEQ ID NO: 35)ACI-36 3A8E12H8* SEQ ID VK_AD GAGGTCCAGCTGCAACAATCTGGACCTGAACTGGTGNO: 46 (SEQ AAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCT and ID NO:TCTGGATATACGTTCACTGACTACTACATGAACTGGG SEQ ID 48/TGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTG NO: 47 SEQ IDGAGATATTAATCCTAACCGTGGTGGAACTACTTACAA NO: 49CCAGAAGTTCAAGGGCAAGGCCACGTTGACTGTAGA andCAAGTCCTCCAGCACAGCCTACATGGAACTCCGCAG SEQ IDCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCA NO: 51)AGTTACTACGCCGTGGGCTACTGGGGCCAAGGCAC CACTCTCACAGTCTCCTCA (SEQ ID NO: 35)ACI-36 3A8E12H8* SEQ ID VK_G GAGGTCCAGCTGCAACAATCTGGACCTGAACTGGTG NO: 46(SEQ AAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCT and ID NO:TCTGGATATACGTTCACTGACTACTACATGAACTGGG SEQ ID 50 andTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTG NO: 47 SEQ IDGAGATATTAATCCTAACCGTGGTGGAACTACTTACAA NO: 51)CCAGAAGTTCAAGGGCAAGGCCACGTTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAACTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGTTACTACGCCGTGGGCTACTGGGGCCAAGGCAC CACTCTCACAGTCTCCTCA (SEQ ID NO: 35)ACI-36 2B6A10C11 SEQ ID SEQ ID GAGGTCCAGCTGCAACAATCTGGACCTGAACTGGTG NO:NO: 50 AAGCCTGGGACTTCAGTGAAGATATCCTGTAAGGCT 46/  andTCTGGATATACGTTCACTGACTACTACATGAACTGGG SEQ SEQ IDTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTG ID NO: NO: 51GAGATATTAATCCTAACCGTGGTGGAACTACTTACAA 52 andCCAGAAGTTTAAGGGCAAGGCCACGTTGACTGTAGA SEQ IDCAAGTCCTCCAGCACAGCCTACATGGAACTCCGCAG NO: 47CCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGTTACTACGCCGTGGGCTACTGGGGCCAAGGCAC CACTCTCACAGTCTCCTCA (SEQ ID NO: 36)ACI-36 2B6G7A12 SEQ ID SEQ ID GAGGTCCAGCTGCAACAATCTGGACCTGAACTGGTG NO:NO: 50 AAGCCTGGGACTTCAGTGAAGATATCCTGTAAGGCT 46/SEQ andTCTGGATATACGTTCACTGACTACTACATGAACTGGG ID NO: SEQ IDTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTG 52 and NO: 51GAGATATTAATCCTAACCGTGGTGGAACTACTTACAA SEQ IDCCAGAAGTTTAAGGGCAAGGCCACGTTGACTGTAGA NO: 47CAAGTCCTCCAGCACAGCCTACATGGAACTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGTTACTACGCCGTGGGCTACTGGGGCCAAGGCAC CACTCTCACAGTCTCCTCA (SEQ ID NO: 36)ACI-36 6H1A11011 SEQ ID SEQ ID GAGGTCCAGCTGCAACAATCTGGACCTGAACTGGTGNO: 46 NO: 50 AAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCT and andTCTGGATACACGTTCACTGACTACTACATGAACTGGG SEQ ID SEQ IDTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTG NO: 47 NO: 51GAGATATTAATCCTAACCGTGGTGGAACTACTTACAACCAGAAGTTCAAGGGCAAGGCCACGTTGACTGTAGACACGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGTTACTACGCCGTGGGCTACTGGGGCCAAGGCAC CACTCTCACAGTCTCCTCA (SEQ ID NO: 37)ACI-36 6H1G6E6 SEQ ID SEQ ID GAGGTCCAGCTGCAACAATCTGGACCTGAACTGGTG NO: 46NO: 50 AAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCT and andTCTGGATACACGTTCACTGACTACTACATGAACTGGG SEQ ID SEQ IDTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTG NO: 47 NO: 51GAGATATTAATCCTAACCGTGGTGGAACTACTTACAACCAGAAGTTCAAGGGCAAGGCCACGTTGACTGTAGACACGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGTTACTACGCCGTGGGCTACTGGGGCCAAGGCAC CACTCTCACAGTCTCCTCA (SEQ ID NO: 37)ACI-33 6C10E5E9C SEQ ID SEQ ID GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGT 12NO: NO:  GAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCACC 53/SEQ 48/CTCTGGATTCACTTTCAGTGACTATGGAATGCACTGG ID SEQ IDGTTCGTCAGGCTCCAGAGAAGGGACTGGAGTGGGT NO: 54 NO: 49TGCATACATTAGTAGTGGCAGTAGTACCATCTACTAT and andGGAGACACAGTGAAGGGCCGATTCACCATCTCCAGA SEQ ID SEQ IDGACAATGCCAAGAACACCCTGTTCCTGCAAATGACC NO: 47 NO: 51AGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGAAGGGGACAGCTCAGGCTACGCCTGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 38) ACI-33 6C10F9C12SEQ ID SEQ ID GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGT A11 NO: NO: 54GAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCACC 53/SEQ andCTCTGGATTCACTTTCAGTGACTATGGAATGCACTGG ID SEQ IDGTTCGTCAGGCTCCAGAGAAGGGACTGGAGTGGGT NO:54 NO: 51TGCATACATTAGTAGTGGCAGTAGTACCATCTACTAT andGGAGACACAGTGAAGGGCCGATTCACCATCTCCAGA SEQ IDGACAATGCCAAGAACACCCTGTTCCTGCAAATGACC NO: 47AGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGAAGGGGACAGCTCAGGCTACGCCTGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 38) ACI-41 7C2(1)F10CSEQ ID SEQ ID GAGGTGAAGCTGATGGAATCTGGAGGAGGCTTGGTA 10D3 NO:  NO:CACCCTGGGGCTTCTCTGAGACTCTACTGTGCAGCT 53/ 49/ TCTGGATTCACCTTTACTGATTACTACATGAGCTGGG SEQ ID SEQ IDTCCGCCAGCCTCCAGGGAAGGCACCTGAGTGGTTG NO: 55 NO: GCTTTGATTAGAAACAAAGCTAATGGTTACACAACAG and 56/AGTATACTGCATCTGTTAAGGGTCGGTTCACCATCTC SEQ ID SEQ IDCAGAGATAATTCCCAAAACATCCTCTATCTTCAAATG NO: 47 NO: 57AACACCCTGAGGGCTGAGGACAGTGCCACTTATTAC andTGTGTAAAAGCTCTGGGACGTTACTTCGATGTCTGG SEQ IDGGCACAGGGACCACGGTCACCGTCTCCTCA  NO: 51 (SEQ ID NO: 39) ACI-41 7C2(2)B9F1SEQ ID SEQ ID GAGGTGAAGCTGATGGAATCTGGAGGAGGCTTGGTA 1D5 NO:  NO: 57CACCCTGGGGCTTCTCTGAGACTCTACTGTGCAGCT 53/ andTCTGGATTCACCTTTACTGATTACTACATGAGCTGGG SEQ ID SEQ IDTCCGCCAGCCTCCAGGGAAGGCACCTGAGTGGTTG NO:55 NO: 51GCTTTGATTAGAAACAAAGCTAATGGTTACACAACAG andAGTATACTGCATCTGTTAAGGGTCGGTTCACCATCTC SEQ IDCAGAGATAATTCCCAAAACATCCTCTATCTTCAAATG NO: 47AACACCCTGAGGGCTGAGGACAGTGCCACTTATTACTGTGTAAAAGCTCTGGGACGTTACTTCGATGTCTGG GGCACAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 39) ACI-35 A4-4A6-18 CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTTCTGAAGCCTGGGGCTTCAGTGAAACTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTG GGTGAAGCAGAGGCCTGGACGAGGCCTTGAGTGGATTGGAAGGATTGATCCTAATAGTGATCGTACTAAGTACAATGAGAAGTTCAAGCGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTATTGTGCAAGGGATGATTACGCCTGGTTTGCTTACTGGGGC CAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 84) ACI-35 A6-1D2-12 CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGTCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGTGAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGGAGTGGCTGGCACACATTTACTGGGATGATGACAAGCGCTATAACGCATCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTCCAGAAACCAGGTATTCCTCAAGATCACCTGTGTGGACACTGCAGATACTGCCACATACTACTGTGCTCGGTTACTGCGTCCTTATGCTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 86) ACI-35 A4-2A1-18GAGGTCCAGCTGCAACAATCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATACACGTTCACTGACTACTACATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTAACAATGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGTCTGACATCTGAGGACTCTGCAGTCTATTATTGTGTAAGAGAGGGGCGGTTTGCTTACTGGGGTCATGGGAC TCTGGTCACTGTCTCTGCA (SEQ ID NO: 109)ACI-35 A4-2A1-40 GAGGTCCAGCTGCAACAATCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATACACGTTCACTGACTACTACATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTAACAATGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGTCTGACATCTGAGGACTCTGCAGTCTATTATTGTGTAAGAGAGGGGCGGTTTGCTTACTGGGGTCATGGGAC TCTGGTCACTGTCTCTGCA (SEQ ID NO: 109)ACI-35 A4-4A6-48 GAGGTCCAGCTGCAACAATCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATACACGTTCACTGACTACTACATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTAACAATGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGTCTGACATCTGAGGACTCTGCAGTCTATTATTGTGTAAGAGAGGGGCGGTTTGCTTACTGGGGTCATGGGAC TCTGGTCACTGTCTCTGCA (SEQ ID NO: 109)ACI-35 A6-2G5-08 CAGGTCCAGCTGAAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAACTGTCCTGCAAGGCTTCTGGCTACACTTTCACTGACTACTATATAAACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGCAAGGATTTATCCTGGAAGAGGTAATATTTACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGAAAAATCCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCTGTCTATTTCTGTGCAAGATTCTGGGACGTGACTTACTGGGGCCAAGG GACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 111) ACI-35 A6-2G5-30 GAGGTCCAGCTGCAACAATCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATTCACGTTCACTGACTACTACATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTAACAATGGTGGTACTAGCTACCACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGTAAGAGAGGGAAGATTTGCTTACTGGGGCCAAGGGACT CTGGTCACTGTCTCTGCA (SEQ ID NO: 113)ACI-35 A6-2G5-41 GAGGTCCAGCTGCAACAATCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATTCACGTTCACTGACTACTACATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTAACAATGGTGGTACTAGCTACCACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGTAAGAGAGGGAAGATTTGCTTACTGGGGCCAAGGGACT CTGGTCACTGTCTCTGCA (SEQ ID NO: 113)Vaccine VK ACI-36 GACATTGTGATGACACAGTCTCCATCCTCCCTGGCTATGTCAGTAGGACAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTGTTTTTAATAGTGGCAATCAAAAGAACTCTTTGGCCTGGTACCAGCAGAAACCAGGACAGTCTCCTAAACTTCTGGTATACTTTGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCATAGGCAGTGGATCTGGGACAGATTTCAGTCTTACCATCAGCAGTGTGCAGGCTGAGGACCTGGCAGATTACTTCTGTCAGGAACATTATACCACTCCTCCCACGTTCGGTACTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO: 40) ACI-36GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGGCTTGTACACAGTCATGGAAAAACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGGTTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGTTCTCAAACTGCACATTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 41) ACI-36GACATTGTGATGACACAGTCTCCATCCTCCCTGGCTATGTCAGTAGGACAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTGTTTTTAATAGTGGCAATCAAAAGAACTCTTTGGCCTGGTACCAGCAGAAACCAGGACAGTCTCCTAAACTTCTGGTATACTTTGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCATAGGCAGTGGATCTGGGACAGATTTCAGTCTTACCATCAGCAGTGTGCAGGCTGAGGACCTGGCAGATTACTTCTGTCAGGAACATTATACCACTCCTCCCACGTTCGGTACTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO: 40) ACI-36GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGGCTTGTACACAGTCATGGAAAAACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGGTTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGTTCTCAAACTGCACATTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 41) ACI-36GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTCATGGAAAAACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGGTTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGTTCTCAAACTGCACATTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 42) ACI-36GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTCATGGAAAAACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGGTTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGTTCTCAAACTGCACATTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 42) ACI-36GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTCTACACAGTCATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGGTTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAACTGCACATTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 43) ACI-36GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTCTACACAGTCATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGGTTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAACTGCACATTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 43) ACI-33GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTCGGCATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATACTACTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGA GCTGAAA (SEQ ID NO: 44) ACI-33GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTCGGCATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATACTACTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGA GCTGAAA (SEQ ID NO: 44) ACI-41GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAGCTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA (SEQ ID NO: 45) ACI-41GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAGCTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA (SEQ ID NO: 45) ACI-35GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAACTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO: 85) ACI-35AACATTTTGATGACACAGTCGCCATCATCTCTGGCTGTGTCTGCAGGAGAAAAGGTCACTATGAGCTGTAAGTCCAGTCAAAGTGTTTTATACAGTTCAAATCAGAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGTGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTTACTCTTACCATCAGCAGTGTACAAGCTGAAGACCTGGCAGTTTATTACTGTCTTCAATACCTCTCCTCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO: 87) ACI-35GATATTGTGATGACTCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCATAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATACATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAAAATCTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 117) ACI-35GATATTR*TGATGACTCAGGCTGCACCCICTGTACCTGICACTCCTGGAGAGTCAGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCATAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAAAATCTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 110) R* = A or G ACI-35GATATTGTGATGACTCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCATAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGGATGICCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAAAATCTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 119) ACI-35GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTTCCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 112) ACI-35GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGA AATAAAA (SEQ ID NO: 114) ACI-35GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGA AATAAAA (SEQ ID NO: 114) *Twoproductive V_(K) sequences (sequences 6 and 7 in Table 10; sequences 40and 41 in Table 11) were isolated from cell lines 3A8A12G7 and 3A8E12H8;the ″VK_G″ sequences were prepared from clones made using ″G″ primer mixand ″V_(K)_AD″ sequences from clones made using ″A″ and ″D″ primermixes. Accordingly, two antibodies with different kappa sequences areproduced by these hybridomas.

TABLE 12 Primers used for CDR sequencing of antibody variable regions AbSEQ ID Subclone isotype Primer sequences NO 3A8A 12G7 IgG2b VH primers5′ GGGAATTCATGRAATGSASCTGGGTYWTYCTCTT 46 3′CCCAAGCTTCCAGGGRCCARKGGATARACIGRTGG 47 VK primers 5′GGGAATTCATGRAGWCACAKWCYCAGGTCTTT 48 ADACTAGTCGACATGGGCWTCAAGATGRAGTCACAKWYYCWGG 49 5′GACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTGGTGCT 50 3′CCCAAGCTTACTGGATGGTGGGAAGATGGA 51 3A8E 12H8 IgG2b VH primers 5′GGGAATTCATGRAATGSASCTGGGTYWTYCTCTT 46 3′CCCAAGCTTCCAGGGRCCARKGGATARACIGRTGG 47 VK primers 5′GGGAATTCATGRAGWCACAKWCYCAGGTCTTT 48 ADACTAGTCGACATGGGCWTCAAGATGRAGTCACAKWYYCWGG 49 5′GACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTGGTGCT 50 3′CCCAAGCTTACTGGATGGTGGGAAGATGGA 51 2B6A 10C11 IgG2b VH primers 5′GGGAATTCATGRAATGSASCTGGGTYWTYCTCTT 46ACTAGTCGACATGGGATGGAGCTRTATCATSYTCTT 52 3′CCCAAGCTTCCAGGGRCCARKGGATARACIGRTGG 47 VK primers 5′ACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTGGTGCT 50 3′CCCAAGCTTACTGGATGGTGGGAAGATGGA 51 2B6G 7A12 IgG2b VH primers 5′GGGAATTCATGRAATGSASCTGGGTYWTYCTCTT 46ACTAGTCGACATGGGATGGAGCTRTATCATSYTCTT 52 3′CCCAAGCTTCCAGGGRCCARKGGATARACIGRTGG 47 VK primers 5′ACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTGGTGCT 50 3′CCCAAGCTTACTGGATGGTGGGAAGATGGA 51 6H1A11C11 IgG2b VH primers 5′GGGAATTCATGRAATGSASCTGGGTYWTYCTCTT 46 3′CCCAAGCTTCCAGGGRCCARKGGATARACIGRTGG 47 VK primers 5′ACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTGGTGCT 50 3′CCCAAGCTTACTGGATGGTGGGAAGATGGA 51 6H1G 6E6 IgG2b VH primers 5′GGGAATTCATGRAATGSASCTGGGTYWTYCTCTT 46 3′CCCAAGCTTCCAGGGRCCARKGGATARACIGRTGG 47 VK primers 5′ACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTGGTGCT 50 3′CCCAAGCTTACTGGATGGTGGGAAGATGGA 51 6C10F9 IgG3 VH primers 5′GGGAATTCATGRASTTSKGGYTMARCTKGRTTT 53 C12A11ACTAGTCGACATGGACTCCAGGCTCAATTTAGTTTTCCT 54 3′CCCAAGCTTCCAGGGRCCARKGGATARACIGRTGG 47 VK primers 5′ACTAGTCGACATGGACTCCAGGCTCAATTTAGTTTTCCT 54 3′CCCAAGCTTACTGGATGGTGGGAAGATGGA 51 6C10E5E IgG3 VH primers 5′GGGAATTCATGRASTTSKGGYTMARCTKGRTTT 53 9C12ACTAGTCGACATGGACTCCAGGCTCAATTTAGTTTTCCT 54 3′CCCAAGCTTCCAGGGRCCARKGGATARACIGRTGG 47 VK primers 5′GGGAATTCATGRAGWCACAKWCYCAGGTCTTT 48ACTAGTCGACATGGGCVVTCAAGATGRAGTCACAKVVYYCWGG 49 3′CCCAAGCTTACTGGATGGTGGGAAGATGGA 51 7C2(1)F10C IgG2b VH primers 5′GGGAATTCATGRASTTSKGGYTMARCTKGRTTT 53 10D3ACTAGTCGACATGAAGVVTGTGGBTRAACTGGRT 55 3′CCCAAGCTTCCAGGGRCCARKGGATARACIGRTGG 47 VK primers 5′ACTAGTCGACATGGAGWCAGACACACISCTGYTATGGGT 56ACTAGTCGACATGGGCVVTCAAGATGRAGTCACAKVVYYCWGG 49ACTAGTCGACATGGTYCTYATVTTRCTGCTGCTATGG 57 3′CCCAAGCTTACTGGATGGTGGGAAGATGGA 51 7C2(2)B9F IgG2b VH primers 5′GGGAATTCATGRASTTSKGGYTMARCTKGRTTT 53 11D5ACTAGTCGACATGAAGWTGTGGBTRAACTGGRT 55 3′CCCAAGCTTCCAGGGRCCARKGGATARACIGRTGG 47 VK primers 5′ACTAGTCGACATGGTYCTYATVTTRCTGCTGCTATGG 57 3′CCCAAGCTTACTGGATGGTGGGAAGATGGA 51 A6-2G5-08 IgG2a VH primers 5′GGGAATTCATGGAATGCAGCTGGGTTTTTCTCTT 120GGGAATTCATGGAATGGAGCTGGGTCTTTCTCTT 121GGGAATTCATGGAATGCAGCTGGGTCATTCTCTT 122GGGAATTCATGGAATGGAGCTGGGTTTTTCTCTT 123GGGAATTCATGGAATGGAGCTGGGTTATTCTCTT 124 GGGAATTCATGAAATGGAGCTGGGTCTTTTCTT125 GGGAATTCATGGAATGCAGCTGGGTCTTCCTCTT 126GGGAATTCATGGAATGGAGCTGGGTTTTCCTCTTC 127 3′CCCAAGCTTCCAGGGACCAATGGATAACGGGTGG 128CCCAAGCTTCCAGGGACCAATGGATAAACGATGG 129CCCAAGCTTCCAGGGACCAATGGATAAACGGTGG 130CCCAAGCTTCCAGGGACCAATGGATAAACGGATGG 131CCCAAGCTTCCAGGGACCAGTGGATAGACGGGTGG 132CCCAAGCTTCCAGGGACCAAGGGATAGATGATGG 133CCCAAGCTTCCAGGGGCCAATGGATAAACGGGTGG 134CCCAAGCTTCCAGGGGCCAATGGATAAACGATGG 135 VK primers 5′ACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTGGTGCT 50 3′CCCAAGCTTACTGGATGGTGGGAAGATGGA 51 A4-2A1-18 IgG2b VH primers 5′GGGAATTCATGGAATGGAGCTGGGTCATTCTCTT 136GGGAATTCATGGAATGCAGCTGGGTTTTTCTCTT 120GGGAATTCATGGAATGGAGCTGGGTTTTTCTCTT 123GGGAATTCATGGAATGCACCTGGGTTTTCCTCTT 137GGGAATTCATGGAATGGAGCTGGGTCTTCCTCTT 138GGGAATTCATGGAATGGAGCTGGGTCATCCTCTT 139GGGAATTCATGGAATGGAGCTGGGTTATTCTCTT 124ACTAGTCGACATGGGATGAGCTTATCATCCTCTT 140 3′CCCAAGCTTCCAGGGGCCAATGGATAACGGTGG 141CCCAAGCTTCCAGGGACCAGTGGGATAAACGGGTGG 142CCCAAGCTTCCAGGGGCCAATGGATAAACGGGTGG 134CCCAAGCTTCCAGGGACCAAGGGATAGACGGGTGG 143CCCAAGCTTCCAGGGACCAAGGGATAAACGGATGG 144CCCAAGCTTCCAGGGACCAGGGGATAAACGGATGG 145CCCAAGCTTCCAGGGACCAATGGATAAACGGATGG 131CCCAAGCTTCCAGGGGCCAGGGATAAACGGGTGG 146CCCAAGCTTCCAGGGGCCAATGGATAAACCGGTGG 147CCCAAGCTTCCAGGGACCAGTGGATAAACGGTGG 148 VK primers 5′ACTAGTCGACATGGTGTCCACAGCTCAGTTCCTTG 149 3′CCCAAGCTTACTGGATGGTGGGAAGATGGA 51 A6-2G5-30 IgG2b VH primers 5′GGGAATTCATGAAATGGAGCTGGGTCTTCCTCTT 150GGGAATTCATGGAATGCAGCTGGGTTATTCTCTT 151GGGAATTTATGGAATGGAGCTGGGTCTTCCTCTT 152GGGAATTCATGGAATGGAGCTGGGTTTTCCTCTT 127GGGAATTCATGGAATGCAGCTGGGTCATCCTCTT 153GGGAATTCATGGAATGGAGCTGGGTTATTCTCTT 124GGGAATTCATGGAATGCAGCTGGGTTTTCCTCTT 154GGGAATTCATGGAATGCAGCTGGGTCTTTCTCTT 155ACTAGTCGACATGGGATGGAGCTATATCATCCTCTT 156ACTAGTCGACATGGGATGGAGCTTATCATCTTCTT 157ACTAGTCGACATGTAGATGTGGTTAAACTGGGT 158 3′CCCAAGCTTCCAGGGGCCAGGGGATAAACGGATGG 159CCCAAGCTTCCAGGGGCCAAGGGATAGACGGATGG 160CCCAAGCTTCCAGGGACCAGGGGATAGACGGGTGG 161CCCAAGCTTCCAGGGACCAGGGGATAGACGGATGG 162CCCAAGCTTCCAGGGGCCAGTGGATAAACGGATGG 163CCCAAGCTTCCAGGGGCCAATGGATAACGATGG 164 CCCAAGCTTCCAGGGGCCAGTGGATAAACGATGG165 CCCAAGCTTCCAGGGACCAATGGATAAACGGTGG 130CCCAAGCTTCCAGGGACCAGTGGATAAACGATGG 166 CCCAAGCTTCCAGGGACCAATGGATAACGATGG167 CCCAAGCTTCCAGGGACCATGGATAAACGGGTGG 168 VK primers 5′ACTAGTCGACATGGGCATCAAGATGAAGTCACATACTCTGG 169ACTAGTCGACATGGGCATCAAGATGAGTCACATACTCTGG 170ACTAGTCGACTGGGCATCAGATGAGTCACATACTCTGG 171ACTAGTCGACATGGGCATCAAGATGAAGTCACAGACCCAGG 172ACTAGTCGACATGGGCTTCAAGATGAAGTCACATTCTCTGG 173ACTAGTCGACATGGGCTTCAAGATGAAGTCACATATTCAGG 174CCCAAGCTTACTGGATGGTGGGAAGATGGA 51 3′ CCCAAGCTTACTGGATGGTGGGAAGATGGA 51A4-2A1-40 IgG2b VH primers 5′ GGGAATTCATGGAATGGAGCTGGGTCATCCTCTT 139GGGAATTCATGGAATGCAGCTGGGTTTTCCTCTT 154GGGAATTCATGGAATGCAGCTGGGTCTTTCTCTT 155GGGAATTCATGGAATGGAGCTGGGTTTTCCTCTT 127GGGAATTCATGGAATGGAGCTGGGTCTTTCTCTT 121ACTAGTCGACATGGATGGAGCTTATCATCCTCTT 175 3′CCCAAGCTTCCAGGGACCAAGGGATAAACGGTGG 176CCCAAGCTTCCAGGGGCCAATGGATAAACCGGTGG 147CCCAAGCTTCCAGGGACCAATGGATAAACGATGG 129CCCAAGCTTCCAGGGGCCAGTGGATAAACGGGTGG 177CCCAAGCTTCCAGGGACCAATGGATAACGGGTGG 128 VK primers 5′ACTAGTCGACATGAGGTACTCGGCTCAGTTCCTGGG 178ACTAGTCGACATGAGGTCCCCGGCTCAGTTCCTGGG 179ACTAGTCGACATGAGGACGTCGATTCAGTTCTTGGG 180 3′CCCAAGCTTACTGGATGGTGGGAAGATGGA 51 A6-2G5-41 IgG2b VH primers 5′GGGAATTCATGGAATGGACCTGGGTCATCCTCTT 181GGGAATTCATGGAATGCAGCTGGGTTTTTCTCTT 120GGGAATTCATGGAATGCAGCTGGGTTATCCTCTT 182GGGAATTCATGGAATGGAGCTGGGTTATTCTCTT 124GGGAATTCATGGAATGCAGCTGGGTCTTCCTCTT 126 GGGAATTCATGAATGGATCTGGGTTATTCTCTT183 3′ CCCAAGCTTCCAGGGACCAGGGGATAAACGGGTGG 184CCCAAGCTTCCAGGGACCAAGGGACGGGTGG 185 CCCAAGCTTCCAGGGACCAATGGATAAACAGATGG186 CCCAAGCTTCCAGGGACCAAGGGATAAACGGATGG 144CCCAAGCTTCCAGGGACCAGGGGATAAACGGATGG 145CCCAAGCTTCCAGGGACCAAGGGATAAACGGGTGG 187 VK primers 5′GGGAATTCATGGAGACACATTCCCAGGTCTTT 188 GGGAATTCATGGAGTCACAGTCTCAGGTCTTT189 ACTAGTCGACATGGGCTTCAAGATGGAGTCACATTTTCAGG 190ACTAGTCGACATGGGCATCAAGATGAAGTCACATATTCAGG 191ACTAGTCGACATGGGCTTCAAGATGAAGTCACATTCTCAGG 192CCCAAGCTTACTGGATGGTGGGAAGATGGA 51 3′ CCCAAGCTTACTGGATGGTGGGAAGATGGA 51A4-4A6-48 IgG2b VH primers 5′ ACTAGTCGACATGGGATGGAGCTTATCATGTTCTT 193ACTAGTCGACATGGGATGGAGCTTATCATGCTCTT 194GGGAATTCATGGAATGCACCTGGGTTTTCCTCTT 137GGGAATTCATGGAATGGACCTGGGTTTTCCTCTT 195GGGAATTCATGGAATGGACCTGGGTCTTTCTCTT 196GGGAATTCATGAAATGGAGCTGGGTTATTCTCTT 197GGGAATTCATGGAATGCAGCTGGGTTATTCTCTT 151GGGAATTCATGGAATGGAGCTGGGTCTTTCTCTT 121 3′CCCAAGCTTCCAGGGGCCAATGGATAGACGATGG 198CCCAAGCTTCCAGGGACCAAGGGATAGACGGATGG 199CCCAAGCTTCCAGGGACCAAGGGATAGACGATGG 200 CCCAAGCTTCCAGGGGCCAATGGATAACGGTGG141 CCCAAGCTTCCAGGGACCAGTGGATAAACGATGG 166CCCAAGCTTCCAGGGACCAATGGATAAACGGATGG 131CCCAAGCTTCCAGGGACCAAGGGATAAACGGATGG 144 VK primers 5′ACTAGTCGACATGATGTACCCGGCTCAGTTTCTGGG 201ACTAGTCGACATGAGGACTTCGATTCAGTTCTTGGG 202ACTAGTCTACATGAAGTTGCCTGTTAGGCTGTTGGTGCT 203ACTAGTCGACATGAAGTTGTCTGTTAGGCTGTTGGTGCT 204ACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTGGTGCT 50 3′CCCAAGCTTACTGGATGGTGGGAAGATGGA 51 A4-4A6-18 IgG2b VH primers 5′ATGGGATGGAGCTRTATCATSYTCTT 205 ATGAAGWTGTGGBTRAACTGGRT 206ATGGRATGGASCKKIRTCTTTMTCT 207 3′ CCAGGGRCCARKGGATARACIGRTGG 208VK primers 5′ ATGGAGACAGACACACTCCTGCTAT 209ATGGAGWCAGACACACTSCTGYTATGGGT 210 ATGAAGTTGCCTGTTAGGCTGTTGGTGCT 211ATGGATTTWCARGTGCAGATTWTCAGCTT 212 ATGGTYCTYATVTCCTTGCTGTTCTGG 213ATGGTYCTYATVTTRCTGCTGCTATGG 214 3′ ACTGGATGGTGGGAAGATGGA 215 A6-1D2-12IgG2a VH primers 5′ ATGAAATGCAGCTGGRTYATSTTCTT 216ATGGRCAGRCTTACWTYYTCATTCCT 217 ATGATGGTGTTAAGTCTTCTGTACCT 218 3′CCAGGGRCCARKGGATARACIGRTGG 208 VK primers 5′ ATGRAGWCACAKWCYCAGGTCTTT219 ATGGAGACAGACACACTCCTGCTAT 209 ATGGAGWCAGACACACTSCTGYTATGGGT 210ATGAGGRCCCCTGCTCAGWTTYTTGGIWTCTT 220 ATGGGCWTCAAGATGRAGTCACAKWYYCWGG 221ATGAAGTTGCCTGTTAGGCTGTTGGTGCT 211 ATGGATTTWCARGTGCAGATTWTCAGCTT 212ATGGTYCTYATVTCCTTGCTGTTCTGG 213 ATGGTYCTYATVTTRCTGCTGCTATGG 214 3′ACTGGATGGTGGGAAGATGGA 215 DegenerateCodons: R = A or G S = C or G D = Aor G or T B = C or G or T Y = C or T M = A or C H = A or C or T K = G orT W = A or T V = A or G or C

TABLE 13 Longest isoform of human Tau (441 aa), also called Tau40Longest isoform of human Tau (441 aa),  MAEPRQEFEV MEDHAGTYGL GDRKDQGGYTalso called Tau40 (SEQ ID NO: 67) MHQDQEGDTD AGLKESPLQT PTEDGSEEPGMicrotubule-associated protein tau isoform SETSDAKSTP TAEDVTAPLV DEGAPGKQAA 2 [Homo sapiens]AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG NCBI Reference Sequence: NP_005901.2HVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPP GQKGQANATR IPAKTPPAPKTPPSSGEPPK SGDRSGYSSP GSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAKSRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIINK KLDLSNVQSK CGSKDNIKHVPGGGSVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNITHVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPVVS GDTSPRHLSN VSSTGSIDMVDSPQLATLAD EVSASLAKQG L (SEQ ID NO: 67)

REFERENCE LIST

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1. An antibody, or a functional part thereof, which antibody recognizesand specifically binds to a phospho-epitope on the mammalian Tau proteinor on a fragment thereof, wherein said antibody or antibody fragment hasa high binding affinity to soluble and insoluble Tau protein, andmodulates soluble and insoluble Tau levels, wherein said antibody orantibody fragment binds to an epitope selected from the group consistingof Tau aa 15-20 comprising a phosphorylated Tyr at position 18 (Y18),Tau aa 405-412 comprising a phosphorylated Ser at position 409 (pS409),Tau aa 405-411 comprising a phosphorylated Ser at position 409 (pS409),Tau aa 208-218 comprising a phosphorylated Thr at position 212 (pT212)and a phosphorylated Ser at position (pS214), Tau aa 393-401 comprisinga phosphorylated Ser at position 396 (pS396), Tau aa 396-401 comprisinga phosphorylated Ser at position 396 (pS396), Tau aa 394-400 comprisinga phosphorylated Ser at position 396 (pS396), Tau aa 402-406 comprisinga phosphorylated Ser at position 404 (pS404), and Tau aa 393-400comprising a phosphorylated Ser at position 396 (pS396).
 2. The antibodyof claim 1 or a functional fragment thereof, wherein said antibody orantibody fragment modulates soluble and insoluble Tau levels in thebrain of a mammal.
 3. The antibody of claim 2 or a functional fragmentthereof, wherein said antibody or antibody fragment modulates solubleand insoluble Tau levels in brain cortex and/or hippocampus. 4.(canceled)
 5. (canceled)
 6. (canceled)
 7. The antibody of claim 1 or afunctional fragment thereof, wherein said antibody or antibody fragmentreduces the levels total soluble tau protein, soluble phosphorylated tauprotein and/or pTau paired helical filaments.
 8. (canceled)
 9. Theantibody of claim 1 or a functional fragment thereof, wherein saidantibody or antibody fragment has a dissociation constant of less than10 nM. 10.-23. (canceled)
 24. The antibody or functional part thereof ofclaim 1, comprising a first binding domain which contains a CDR1 withthe amino acid sequence shown in SEQ ID NO: 21, 24, 27, 28, 29, 32, 73,81, 93, 101, 106, or an amino acid sequence at least 70%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% identical thereto; a CDR2 with the amino acid sequenceshown in SEQ ID NO: 22, 25, 30, 33, 74, 82, 94, 102, 107, or an aminoacid sequence at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% identical thereto and a CDR3 with the amino acidsequence shown in SEQ ID NO: 23, 26, 31, 34, 75, 83, 95, 103, 108, or anamino acid sequence at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% or 100% identical thereto; and/or a second binding domain whichcontains in sequence a CDR1 with the amino acid sequence shown in SEQ IDNO: 12, 15, 18, 70, 78, 89, 98, or an amino acid sequence at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% identicalthereto; a CDR2 with the amino acid sequence shown in SEQ ID NO: 13, 16,19, 71, 79, 90, 99, 115, or an amino acid sequence at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% identical thereto and a CDR3 with the amino acidsequence shown in SEQ ID NO: 14, 17, 20, 72, 80, 91, 100, or an aminoacid sequence at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% identical thereto. 25.-42. (canceled)
 43. The antibody orfunctional part thereof of claim 1 comprising a first binding domainwhich contains the amino acid sequence shown in SEQ ID NO: 6, 7, 8, 9,10, 11, or an amino acid sequence at least 90% identical thereto, and/ora second binding domain which contains the amino acid sequence shown inSEQ ID NO: 1, 2, 3, 4, 5, or an amino acid sequence at least 85%identical thereto.
 44. (canceled)
 45. (canceled)
 46. The antibody orfunctional part thereof of claim 1 comprising a first binding domainwhich contains the amino acid sequence shown in SEQ ID NO: 69, 77, 116,92, 97, 105, or an amino acid sequence at least 60%, at least 70%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% identical thereto, and/or a secondbinding domain which contains the amino acid sequence shown in SEQ IDNO: 68, 76, 88, 96, 104, or an amino acid sequence at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% identical thereto. 47.-52.(canceled)
 53. The antibody or functional part thereof of claim 1, whichis polyclonal antibody, a monoclonal antibody, a chimeric antibody, ahumanized antibody or a fully human antibody or a functional fragmentthereof.
 54. (canceled)
 55. (canceled)
 56. The antibody or functionalpart thereof of claim 1, wherein said antibody or functional partthereof recognizes and specifically binds to a phospho-epitope on themammalian Tau protein or on a fragment thereof, but does not bind to thecorresponding unphosphorylated epitope and/or to non-related epitopes.57. A polynucleotide encoding the antibody or functional part thereof ofclaim
 1. 58. The polynucleotide of claim 57 comprising a nucleic acidmolecule selected from the group consisting of a. a nucleic acidmolecule comprising a nucleotide sequence encoding a polypeptidecomprising the amino acid sequence as depicted in SEQ ID NOs: 35-45; b.a nucleic acid molecule comprising a nucleotide sequence that has atleast 85% sequence identity to the sequence shown in SEQ ID NOs: 35-45;SEQ ID NOs: 84-87, SEQ ID NO: 109-112 and 117-119; c. a nucleic acidmolecule comprising a nucleotide sequence that has at least 90% sequenceidentity to the sequence shown in SEQ ID NOs: 35-45; SEQ ID NOs: 84-87,SEQ ID NO: 109-112 and 117; d. a nucleic acid molecule comprising anucleotide sequence that has at least 95% sequence identity to thesequence shown in SEQ ID NOs: 35-45; SEQ ID NOs: 84-87, SEQ ID NO:109-112 and 117-119; e. a nucleic acid molecule comprising a nucleotidesequence that has at least 98% sequence identity to the sequence shownin SEQ ID NOs: 35-45; SEQ ID NOs: 84-87, SEQ ID NO: 109-112 and 117-119;f. a nucleic acid molecule comprising a nucleotide sequence thecomplementary strand of which hybridizes to the nuclei acid molecule ofany of a)-f); g. a nucleic acid molecule comprising a nucleotidesequence that deviates from the nucleotide sequence defined in any ofa)-g) by the degeneracy of the genetic code: wherein said nucleic acidmolecule as defined in any of a)-g) recognizes and specifically binds toa phospho-epitope on a mammalian, particularly on the human Tau proteinor on a fragment thereof, particularly to a pathological protein tauconformer, but does not bind to the corresponding unphosphorylatedepitope and/or to non-related epitopes, wherein said antibody has a highbinding affinity to aggregated Tau protein, and modulates soluble andinsoluble Tau levels.
 59. A pharmaceutical composition comprising theantibody or a functional fragment thereof of claim 1 in atherapeutically effective amount together with a pharmaceuticallyacceptable carrier. 60.-67. (canceled)
 68. A method for the treatment ofa neurodegenerative disease or disorder such as tauopathy comprisingadministering to a mammal suffering from such a disease or disorder, anantibody or a functional fragment thereof of claim
 1. 69. The method ofclaim 68, wherein the neurodegenerative disease or disorder is selectedfrom Alzheimer's Disease, Creutzfeldt-Jacob disease, Dementiapugilistica, Down's Syndrome, Gerstmann-Sträussler-Scheinker disease,inclusion-body myositis, and prion protein cerebral amyloid angiopathy,traumatic brain injury and further diseases or disorders which do notshow a distinct amyloid pathology including, but not limited to,amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam,Non-Guamanian motor neuron disease with neurofibrillary tangles,argyrophilic grain dementia, corticobasal degeneration, diffuseneurofibrillary tangles with calcification, frontotemporal dementia withparkinsonism linked to chromosome 17, Hallevorden-Spatz disease,multiple system atrophy, Niemann-Pick disease, type C,Pallido-ponto-nigral degeneration, Pick's disease, progressivesubcortical gliosis, progressive supranuclear palsy, Subacute sclerosingpanencephalitis Tangle only dementia, Postencephalitic Parkinsonism,Myotonic dystrophy.
 70. A method for inducing a passive immune responsein a mammal suffering from a neurodegenerative disorder comprisingadministering to said mammal an antibody or a functional fragmentthereof of claim
 1. 71. A method of diagnosing a tau-protein-associateddisease, disorder or condition in a patient comprising detecting theimmunospecific binding of an antibody or an active fragment thereof toan epitope of the tau protein in a sample or in situ which includes thesteps of a. bringing the sample or a specific body part or body areasuspected to contain the tau protein into contact with an antibody or afragment thereof according to claim 1, which peptide or fragment thereofbinds an epitope of the tau protein; b. allowing the antibody to bind tothe tau protein to form an immunological complex; c. detecting theformation of the immunological complex; and d. correlating the presenceor absence of the immunological complex with the presence or absence oftau protein in the sample or specific body part or area.
 72. A methodfor diagnosing a predisposition to tau-protein-associated disease,disorder or condition in a patient comprising detecting theimmunospecific binding of an antibody or an active fragment thereof toan epitope of the tau protein in a sample or in situ which includes thesteps of a. bringing the sample or a specific body part or body areasuspected to contain the tau antigen into contact with an antibody or afragment thereof according to claim 1, which peptide or fragment thereofbinds an epitope of the tau protein; b. allowing the antibody to bind tothe tau antigen to form an immunological complex; c. detecting theformation of the immunological complex; and d. correlating the presenceor absence of the immunological complex with the presence or absence oftau antigen in the sample or specific body part or area, e. comparingthe amount of said immunological complex to a normal control value,wherein an increase in the amount of said aggregate compared to a normalcontrol value indicates that said patient is suffering from or is atrisk of developing an tau protein-associated disease or condition.
 73. Amethod for monitoring minimal residual disease in a patient followingtreatment with an antibody or a pharmaceutical composition according toany one of the preceding claims, wherein said method comprises: a.bringing the sample or a specific body part or body area suspected tocontain the tau antigen into contact with an antibody or a fragmentthereof according to claim 1, which peptide or fragment thereof binds anepitope of the tau protein; b. allowing the antibody to bind to the tauantigen to form an immunological complex; c. detecting the formation ofthe immunological complex; and d. correlating the presence or absence ofthe immunological complex with the presence or absence of tau antigen inthe sample or specific body part or area, e. comparing the amount ofsaid immunological complex to a normal control value, wherein anincrease in the amount of said aggregate compared to a normal controlvalue indicates that said patient still suffers from a minimal residualdisease.
 74. A method for predicting responsiveness of a patient beingtreated with an antibody or a pharmaceutical composition according toany one of the preceding claims comprising a. bringing the sample or aspecific body part or body area suspected to contain the tau antigeninto contact with an antibody or a fragment thereof according to claim1, which peptide or fragment thereof binds an epitope of the tauprotein; b. allowing the antibody to bind to the tau antigen to form animmunological complex; c. detecting the formation of the immunologicalcomplex; and d. correlating the presence or absence of the immunologicalcomplex with the presence or absence of tau antigen in the sample orspecific body part or area, e. comparing the amount of saidimmunological complex before and after onset of the treatment, whereinan decrease in the amount of said aggregate indicates that said patienthas a high potential of being responsive to the treatment.
 75. A testkit for detection and diagnosis of tau protein-associated diseases,disorders or conditions comprising an antibody or functional partthereof according to claim
 1. 76. (canceled)
 77. (canceled)
 78. A cellline producing an antibody according to claim
 1. 79.-107. (canceled)108. A cell line comprising the polynucleotide of claim
 57. 109. Amethod of producing an antibody, comprising the step of culturing thecell line of claim 108 in a suitable cultivation medium.
 110. (canceled)111. The method of claim 109, further comprising the step of purifyingthe antibody from the cell line or cultivation medium. 112.-114.(canceled)
 115. A cell line according to claim 78, which is hybridomacell line A4-4A6-48 deposited on Aug. 30, 2011 as DSM ACC3136; orhybridoma cell line A6-2G5-30 deposited on Aug. 30, 2011 as DSM ACC3137;or hybridoma cell line A6-2G5-41 deposited on Aug. 30, 2011 as DSMACC3138; or hybridoma cell line A4-2A1-18 deposited on Aug. 30, 2011 asDSM ACC3139; or hybridoma cell line A4-2A1-40 deposited on Aug. 30, 2011as DSM ACC3140; or hybridoma cell line A6-1D2-12 deposited on Sep. 6,2011 as DSM ACC3141, or hybridoma cell line 6C10F9C12A11 deposited onAug. 25, 2010 as DSM ACC3079, or hybridoma cell line 6C10E5E9C12deposited on Aug. 25, 2010 as DSM ACC3081, or hybridoma cell line6H1A11C11 deposited on Aug. 25, 2010 as DSM ACC3080, or hybridoma cellline 6H1G6E6 deposited on Aug. 25, 2010 as DSM ACC3088, or hybridomacell line 2B6A10C11 deposited on Aug. 25, 2010 as DSM ACC3084, orhybridoma cell line 2B6G7A12 deposited on Mar. 10, 2010 as DSM ACC3087,or hybridoma cell line 3A8A12G7 deposited on Aug. 25, 2010 as DSMACC3086, or hybridoma cell line 3A8E12H8 deposited on Aug. 25, 2010 asDSM ACC3085, or hybridoma cell line 7C2(1)F10C10D3 deposited on Aug. 25,2010 as DSM ACC3082, or hybridoma cell line 7C2(2)B9F11D5 deposited onAug. 25, 2010 as DSM ACC3083. 116.-120. (canceled)